Methods and compositions for genomic integration

ABSTRACT

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

CROSS REFERENCE

This application is continuation of U.S. application Ser. No. 17/499,232 filed on Oct. 12, 2021, which is a continuation in part of and claims priority to International Application No. PCT/US2020/049240, filed Sep. 3, 2020, which claims priority to U.S. Provisional Application No. 62/895,441, filed on Sep. 3, 2019, U.S. Provisional Application No. 62/908,800, filed on Oct. 1, 2019, and U.S. Provisional Application No. 63/039,261, filed on Jun. 15, 2020, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 13, 2020, is named 56371701601_SL.txt and is 363,857 bytes in size.

BACKGROUND

Cell therapy is a rapidly developing field for addressing difficult to treat diseases, such as cancer, persistent infections and certain diseases that are refractory to other forms of treatment. Cell therapy often utilizes cells that are engineered ex vivo and administered to an organism to correct deficiencies within the body. An effective and reliable system for manipulation of a cell's genome is crucial, in the sense that when the engineered cell is administered into an organism, it functions optimally and with prolonged efficacy. Likewise, reliable mechanisms of genetic manipulation form the cornerstone in the success of gene therapy. However, severe deficiencies exist in methods for delivering nucleic acid cargo (e.g., large cargo) in a therapeutically safe and effective manner. Viral delivery mechanisms are frequently used to deliver large nucleic acid cargo in a cell but are tied to safety issues and cannot be used to express the cargo in some cell types. Additionally, subjecting a cell to repeated gene manipulation can affect cell health, induce alterations of cell cycle and render the cell unsuitable for therapeutic use. Advancements are continually sought in the area for efficacious delivery and stabilization of an exogenously introduced genetic material for therapeutic purposes.

SUMMARY

Provided herein are compositions and methods for stable, non-viral transfer and integration of genetic material into a cell. In one aspect, the genetic material is a self-integrating polynucleotide. The genetic material can be stably integrated in the genome of the cell. The cell may be a human cell. The method is designed for a safe and reliable integration of a genetic material into the genome of a cell.

Provided herein is pharmaceutical composition comprising a therapeutically effective amount of one or more polynucleic acids, or at least one vector encoding the one or more polynucleic acids, the one or more polynucleic acids comprising: (a) a mobile genetic element comprising a sequence encoding a polypeptide; and (b) an insert sequence, wherein the insert sequence comprises a sequence that is a reverse complement of a sequence encoding an exogenous therapeutic polypeptide, wherein the polypeptide encoded by the sequence of the mobile genetic element promotes integration of the insert sequence into a genome of a cell; and wherein the pharmaceutical composition is substantially non-immunogenic to a human subject.

In some embodiments, the polypeptide encoded by the sequence of the mobile genetic element comprises one or more long interspersed nuclear element (LINE) polypeptides, wherein the one or more LINE polypeptides comprises: (i) human ORF1p or a functional fragment thereof, and (ii) human ORF2p or a functional fragment thereof.

In some embodiments, the insert sequence stably integrates and/or is retrotransposed into the genome of a human cell.

In some embodiments, the human cell is an immune cell selected from the group consisting of a T cell, a B cell, a myeloid cell, a monocyte, a macrophage and a dendritic cell.

In some embodiments, the insert sequence is integrated into the genome (i) by cleavage of a DNA strand of a target site by an endonuclease encoded by the one or more polynucleic acids, (ii) via target-primed reverse transcription (TPRT) or (iii) via reverse splicing of the insert sequence into a DNA target site of the genome.

In some embodiments, the insert sequence is integrated into the genome at a poly T site using specificity of an endonuclease domain of the human ORF2p.

In some embodiments, the poly T site comprises the sequence TTTTTA.

In some embodiments, the one or more polynucleic acids comprises homology arms complementary to a target site in the genome.

In some embodiments, the insert sequence integrates into: (a) the genome at a locus that is not a ribosomal locus; (b) a gene or regulatory region of a gene of the genome, thereby disrupting the gene or downregulating expression of the gene; (c) a gene or regulatory region of a gene of the genome, thereby upregulating expression of the gene; or (d) the genome and replaces a gene of the genome.

In some embodiments, the pharmaceutical composition further comprises (i) one or more siRNAs and/or (ii) an RNA guide sequence or a polynucleic acid encoding the RNA guide sequence, and wherein the RNA guide sequence targets a DNA target site of the genome and the insert sequence is integrated into the genome at the DNA target site of the genome.

In some embodiments, the one or more polynucleic acids have a total length of from 3 kb to 20 kb.

In some embodiments, the one or more polynucleic acids comprises one or more polyribonucleic acids, one or more RNAs or one or more mRNAs.

In some embodiments, the exogenous therapeutic polypeptide is selected from the group consisting of a ligand, an antibody, a receptor, an enzyme, a transport protein, a structural protein, a hormone, a contractile protein, a storage protein and a transcription factor.

In some embodiments, the exogenous therapeutic polypeptide is a receptor selected from the group consisting of a chimeric antigen receptor (CAR) and a T cell receptor (TCR).

In some embodiments, the one or more polynucleic acids comprises a first expression cassette comprising a promoter sequence, a 5′ UTR sequence, a 3′ UTR sequence and a poly A sequence; wherein: (i) the promoter sequence is upstream of the 5′ UTR sequence, (ii) the 5′ UTR sequence is upstream of the sequence of the mobile genetic element encoding a polypeptide, (iii) the 3′ UTR sequence is downstream of the insert sequence; and (iv) the 3′ UTR is upstream of the poly A sequence; and wherein the 5′ UTR sequence, the 3′ UTR sequence or the poly A sequence comprises a binding site for a human ORF2p or a functional fragment thereof.

In some embodiments, the insert sequence comprises a second expression cassette comprising a sequence that is a reverse complement of a promoter sequence, a sequence that is a reverse complement of a 5′ UTR sequence, a sequence that is a reverse complement of a 3′ UTR sequence and a sequence that is a reverse complement of a poly A sequence; wherein: (i) the sequence that is a reverse complement of a promoter sequence is downstream of the sequence that is a reverse complement of a 5′ UTR sequence, (ii) the sequence that is a reverse complement of a 5′ UTR sequence is downstream of the sequence that is a reverse complement of a sequence encoding an exogenous therapeutic polypeptide (iii) the sequence that is a reverse complement of a 3′ UTR sequence is upstream of the sequence that is a reverse complement of a sequence encoding an exogenous therapeutic polypeptide, and (iv) the sequence that is a reverse complement of a poly A sequence is upstream of the sequence that is a reverse complement of a 3′ UTR sequence and downstream of the sequence of the mobile genetic encoding a polypeptide.

In some embodiments, the promoter sequence of the first expression cassette is different from the promoter sequence of the second expression cassette.

In some embodiments, the one or more LINE polypeptides comprises a first LINE polypeptide comprising the human ORF1p or functional fragment thereof and a second LINE polypeptide comprising the human ORF2p or functional fragment thereof, wherein the first LINE polypeptide and the second LINE polypeptide are translated from different open reading frames (ORFs).

In some embodiments, the one or more polynucleic acids comprises a first polynucleic acid molecule encoding the human ORF1p or functional fragment thereof and a second polynucleic acid molecule encoding the human ORF2p or functional fragment thereof.

In some embodiments, the one or more polynucleic acids comprises a 5′ UTR sequence and a 3′ UTR sequence, wherein (a) the 5′ UTR comprises a 5′ UTR from LINE-1 or a sequence with at least 80% sequence identity to ACUCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCUGACCCUGCACUGUCCCAGCACC; and/or (b) the 3′ UTR comprises a 3′ UTR from LINE-1 or a sequence with at least 80% sequence identity to

CAGGACACAGCCUUGGAUCAGGACAGAGACUUGGGGGCCAUCCUGCCCC UCCAACCCGACAUGUGUACCUCAGCUUUUUCCCUCACUUGCAUCAAUAA AGCUUCUGUGUUUGGAACAG.

In some embodiments, the sequence encoding the exogenous therapeutic polypeptide does not comprise introns.

In some embodiments, the polypeptide encoded by the sequence of the mobile genetic element comprises a C-terminal nuclear localization signal (NLS), an N-terminal NLS or both.

In some embodiments, the sequence encoding the exogenous polypeptide is not in frame with a sequence encoding the ORF1p or functional fragment thereof and/or is not in frame with a sequence encoding the ORF2p or functional fragment thereof.

In some embodiments, the one or more polynucleic acids comprises a sequence encoding a nuclease domain, a nuclease domain that is not derived from ORF2p, a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a Cas6 domain, a Cas7 domain, a Cas8 domain, a zinc finger binding domain from an R2 retroelement, or a DNA binding domain that binds to repeat sequences.

In some embodiments, the one or more polynucleic acids comprises a sequence encoding the nuclease domain, wherein the nuclease domain does not have nuclease activity or comprises a mutation that reduces activity of the nuclease domain compared to the nuclease domain without the mutation.

In some embodiments, the ORF2p or functional fragment thereof lacks endonuclease activity or comprises a mutation selected from the group consisting of S228P and Y1180A, and/or wherein the ORF1p or functional fragment comprises a K3R mutation.

In some embodiments, the insert sequence comprises a sequence that is a reverse complement of a sequence encoding two or more exogenous therapeutic polypeptides.

In some embodiments, the one or more polynucleic acids comprises one or more polyribonucleic acids, wherein the exogenous therapeutic polypeptide is a receptor selected from the group consisting of a chimeric antigen receptor (CAR) and a T cell receptor (TCR), and wherein the pharmaceutical composition is formulated for systemic administration to a human subject.

In some embodiments, the one or more polynucleic acids (i) are formulated in a nanoparticle selected from the group consisting of a lipid nanoparticle and a polymeric nanoparticle; and/or (ii) comprises one or more polynucleic acids selected from the group consisting of glycosylated RNAs, circular RNAs and self-replicating RNAs.

Also provided herein is a method of treating a disease or condition in a human subject in need thereof comprising administering a pharmaceutical composition described herein to the human subject.

Also provided herein is a method of modifying a population of human cells ex vivo comprising contacting a composition to a population of human cell ex vivo, thereby forming an ex vivo modified population of human cells, the composition comprising one or more polynucleic acids, or at least one vector encoding the one or more polynucleic acids, the one or more polynucleic acids comprising: (a) a mobile genetic element comprising a sequence encoding a polypeptide; and (b) an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous therapeutic polypeptide, wherein the ex vivo modified population of human cells is substantially non-immunogenic to a human subject.

In one aspect, provided herein are compositions and methods that allow integration of genetic material into the genome of a cell, wherein the genetic material that can be integrated is not specifically restricted by size. In some aspects, the method described herein provides a one-step, single polynucleotide-mediated delivery and integration of genetic “cargo” in the genome of a cell. The genetic material may comprise a coding sequence, e.g., a sequence encoding a transgene, a peptide, a recombinant protein, or an antibody or fragments thereof, wherein the method and compositions ensure stable expression of the transcribed product encoded by the coding sequence. The genetic material may comprise a non-coding sequence, for example, a regulatory RNA sequences, e.g., a regulatory small inhibitory RNA (siRNA), microRNA (miRNA), long non-coding RNA (lncRNA), or one or more transcription regulators such as a promoter and/or an enhancer, and may also include, but not limited to structural biomolecules such as ribosomal RNA (rRNA), transfer RNA (tRNA) or a fragment thereof or a combination thereof.

In another aspect, provided herein are methods and compositions for site-specific integration of a genetic material that may not be specifically restricted by size, into the genome of a cell via a non-viral delivery that ensures both safety and efficacy of the transfer. Provided methods and compositions may be particularly useful in developing a therapeutic, such as a therapeutic comprising a polynucleotide comprising a genetic material and a machinery that allows transfer into a cell and stable integration into the genome of the cell into which the polynucleotide or an mRNA encoding the polynucleotide is transferred. In some embodiments, the therapeutic may be a cell that comprises a polynucleotide that has been stably integrated into the genome of the cell using the methods and compositions described herein.

In one aspect, the present disclosure provides compositions and methods for stable gene transfer into a cell. In some embodiments, the compositions and methods are for stable gene transfer into an immune cell. In some cases, the immune cell is a myeloid cell. In some cases, the methods described herein relate to development of myeloid cells for immunotherapy.

Provided herein is a method of treating a disease in a subject in need thereof, comprising: administering a pharmaceutical composition to the subject wherein the pharmaceutical composition comprises a polycistronic mRNA sequence encoding a gene or fragment thereof, operably linked to a sequence encoding an L1 retrotransposon; wherein the gene or the fragment thereof is at least 10.1 kb in length.

Provided herein is a method for integrating a nucleic acid sequence into the genome of a cell, comprising contacting the cell with a composition comprising a polycistronic mRNA sequence encoding a gene or fragment thereof, operably linked to a sequence encoding an L1 retrotransposon; wherein the gene or the fragment thereof is at least 10.1 kb in length. In some embodiments, the gene or the fragment thereof (e.g., the payload) is at least about 10.2 kb, 10.3 kb, 10.4 kb, 10.5 kb, 10.6 kb, 10.7 kb, 10.8 kb, 10.9 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb or more in length.

Provided herein is a method for integrating a nucleic acid sequence into the genome of a cell, comprising contacting the cell with a composition comprising a polycistronic mRNA sequence encoding a gene or fragment thereof, operably linked to a sequence encoding an L1 retrotransposon; wherein the gene or the fragment thereof is selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF, and F8.

Provided herein is a method of expressing a protein encoded by a recombinant nucleic acid in a cell, the method comprising integrating a nucleic acid sequence into the genome of a cell by contacting the cell with a composition comprising a polycistronic mRNA sequence encoding a gene or fragment thereof, operably linked to a sequence encoding an L1 retrotransposon; and expressing a protein encoded by the gene or fragment thereof, wherein expression of the protein is detectable more than 30 days after (a).

In one embodiment of a method described herein, the disease is a genetic disease.

Provided herein is a method of treating Stargardt disease, LCA10, USH1D, DFNB12, retinitis pigmentosa (RP) USH2A, USH2C, Alstrom syndrome, Glycogen storage disease III, Non-syndromic deafness, Hemophilia A, or Leber congenital amaurosis in a subject, the method comprising: (i) introducing into the subject an mRNA encoding a suitable gene or a fragment thereof, operably linked to a human L1 transposon, or (ii) introducing to the subject a population of cells comprising an mRNA encoding a suitable gene or a fragment thereof, operably linked to a human L1 transposon.

In one embodiment of a method described herein, the method comprises treating Stargardt disease in a subject in need thereof, and wherein the mRNA encodes an ABCA4 gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating Usher Syndrome Type 1b (Usher 1b) disease in a subject in need thereof, and wherein the mRNA encodes an MY07A gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating Leber congenital amaurosis (LCA)10 disease in a subject in need thereof, and wherein the mRNA encodes a CEP290 gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating a User Syndrome Type 1D (USH1D) non-syndromic deafness or hearing loss USH1D, DFN12 disease in a subject in need thereof, and wherein the mRNA encodes a CDH23 gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating a retinitis pigmentose (RP) disease in a subject in need thereof, and wherein the mRNA encodes an EYS gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating a User Syndrome Type 2A (USH2A) and wherein the mRNA encodes an USH2a gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating a User Syndrome Type 2C (USH2C) and wherein the mRNA encodes a GPR98 gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating an Altrom Syndrome, and wherein the mRNA encodes an ALMS1 gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating a Glycogen Storage Disease III, and wherein the mRNA encodes a GDE gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating a non-syndromic deafness or hearing loss and wherein the mRNA encodes an OTOF gene, or a fragment thereof.

In one embodiment of a method described herein, the method comprises treating Hemophilia A, and the mRNA encodes an Factor VIII (F8) gene, or a fragment thereof.

Provided herein is a method for targeted replacement of a genomic nucleic acid sequence of a cell, the method comprising: (A) introducing to the cell a polynucleotide sequence encoding a first protein complex comprising a targeted excision machinery for excising from the genome of the cell a nucleic acid sequence comprising one or more mutations; and (B) a recombinant mRNA encoding a second protein complex, wherein the recombinant mRNA comprises: (i) a nucleic acid sequence comprising the excised nucleic acid sequence in (A) that does not contain the one or more mutations, and (ii) a sequence encoding an L1 retrotransposon ORF2 protein under the influence of an independent promoter.

In one embodiment of a method described herein, the nucleic acid sequence comprising the one or more mutations comprises a pathogenic variant of a cellular gene.

In one embodiment of a method described herein, the a nucleic acid sequence in (B) comprising the nucleic acid sequence that does not contain the one or more mutations is operably linked to the ORF2 sequence.

In one embodiment of a method described herein, the method further comprising introducing a sequence comprising a plurality of thymidine residues at the excision site.

In some embodiment, introducing the sequence comprises introducing at least four thymidine residues.

In one embodiment of a method described herein, the targeted excision machinery comprises a sequence guided site-specific excision endonuclease.

In one embodiment of a method described herein, the targeted excision machinery comprises a CRISPR-CAS system.

In some embodiments, the targeted excision machinery is a modified recombinant LINE 1 (L1) endonuclease.

In some embodiments, introducing the sequence comprising a plurality of thymidine residues comprises base extension by prime editing at the excision site.

In some embodiments, the mRNA sequence encoding an L1 retrotransposon ORF2 protein further comprises a sequence encoding the L1 retrotransposon ORF1 protein.

In some embodiments, the mRNA comprises a sequence for an inducible promoter.

In one embodiment of a method described herein, the excised sequence is greater than 1000 bases.

In one embodiment of a method described herein, the excised sequence is greater than 6 kb.

In one embodiment of a method described herein, the excised sequence is about 10 kb.

In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is an epithelial cell. In some embodiments, the cell is a cancer cell.

In some embodiments, the nucleic acid sequence encodes an ATP-binding cassette (ABC) transporter gene, (ABCA4) gene, or a fragment thereof.

In some embodiments, the nucleic acid sequence encodes an MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF or an F8 gene or a fragment thereof.

In some embodiments, introducing comprises introducing to the cell ex vivo. In some embodiments, introducing comprises electroporation. In some embodiments, introducing comprises introducing to the cell in vivo. In some embodiments, expression of the nucleic acid sequence comprising the sequence that does not contain the one or more mutations, is detectable at least 35 days after introducing to the cell. In some embodiments, introducing into the subject comprises direct administration of the mRNA systemically.

In some embodiments, introducing into the subject comprises local administration of the mRNA.

In some embodiments, the mRNA sequence comprises a cell targeting moiety.

In some embodiments, the cell targeting moiety is an aptamer.

In some embodiments, introducing into the subject comprises introducing the mRNA in the retina of the subject.

Provided herein is a method of integrating a nucleic acid sequence into a genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA into the cell, wherein the mRNA comprises: (a) an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence, or (ii) a sequence that is a reverse complement of the exogenous sequence; (b) a 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for a human ORF protein, and wherein the insert sequence is integrated into the genome of the cell, wherein the insert sequence is a gene selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF, and F8.

In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for human ORF2p.

Provided herein is a method for integrating a nucleic acid sequence into the genome of an immune cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises: (a) an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; (b) 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence, wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and wherein the insert sequence is integrated into the genome of the immune cell, wherein the insert sequence is a gene selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF, and F8.

Provided herein is a method for integrating a nucleic acid sequence into the genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises: (a) an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; (b) a 5′ UTR sequence, a sequence of a human retrotransposon downstream of the 5′ UTR sequence, and a 3′ UTR sequence downstream of the sequence of a human retrotransposon; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and wherein the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs, and wherein the insert sequence is integrated into the genome of the cell, wherein the insert sequence is a gene selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF, and F8.

In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises an ORF2p binding site. In some embodiments, the ORF2p binding site is a poly A sequence in the 3′ UTR sequence.

In some embodiments, the mRNA comprises a sequence of a human retrotransposon. In some embodiments, the sequence of a human retrotransposon is downstream of the 5′ UTR sequence.

In some embodiments, the sequence of a human retrotransposon is upstream of the 3′ UTR sequence. In some embodiments, the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs. In some embodiments, the two ORFs are non-overlapping ORFs.

In some embodiments, the sequence of a human retrotransposon comprises a sequence of a non-LTR retrotransposon. In some embodiments, the sequence of a human retrotransposon encodes comprises a LINE-1 retrotransposon. In some embodiments, the LINE-1 retrotransposon is a human LINE-1 retrotransposon. In some embodiments, the sequence of a human retrotransposon comprises a sequence encoding an endonuclease and/or a reverse transcriptase.

In some embodiments, the endonuclease and/or a reverse transcriptase is ORF2p.

In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase domain.

In some embodiments, the endonuclease and/or a reverse transcriptase is a minke whale endonuclease and/or a reverse transcriptase.

In some embodiments, the sequence of a human retrotransposon comprises a sequence encoding ORF2p. In some embodiments, the insert sequence is integrated into the genome at a poly T site using specificity of an endonuclease domain of the ORF2p. In some embodiments, the poly T site comprises the sequence TTTTTA. In some embodiments, the retrotransposon comprises an ORF1p and/or the ORF2p fused to a nuclear retention sequence. In some embodiments, the nuclear retention sequence is an Alu sequence. In some embodiments, the ORF1p and/or the ORF2p is fused to an MS2 coat protein. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises at least one, two, three or more MS2 hairpin sequences.

Provided herein is a composition comprising a recombinant mRNA or vector encoding an mRNA, wherein the mRNA comprises a human LINE-1 transposon sequence comprising: (i) a human LINE-1 transposon 5′ UTR sequence, (ii) a sequence encoding ORF1p downstream of the human LINE-1 transposon 5′ UTR sequence, (iii) an inter-ORF linker sequence downstream of the sequence encoding ORF1p, (iv) a sequence encoding ORF2p downstream of the inter-ORF linker sequence, and (v) a 3′ UTR sequence derived from a human LINE-1 transposon downstream of the sequence encoding ORF2p; wherein the 3′ UTR sequence comprises an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element, wherein the insert sequence is a gene selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF, and F8.

Provided herein is a composition comprising a nucleic acid comprising a nucleotide sequence encoding (a) a long interspersed nuclear element (LINE) polypeptide, wherein the LINE polypeptide includes human ORF1p and human ORF2p; and (b) an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element, wherein the composition is substantially non-immunogenic, wherein the insert sequence is a gene selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF, and F8.

Immunotherapy using phagocytic cells involves making and using engineered myeloid cells, such as macrophages or other phagocytic cells that attack and kill diseased cells, such as cancer cells, or infected cells. Engineered myeloid cells, such as macrophages and other phagocytic cells are prepared by incorporating in them via recombinant nucleic acid technology, a synthetic, recombinant nucleic acid encoding an engineered protein, such as a chimeric antigen receptor, that comprises a targeted antigen binding extracellular domain that is designed to bind to specific antigens on the surface of a target, such as a target cell, such as a cancer cell. Binding of the engineered chimeric receptor to an antigen on a target, such as cancer antigen (or likewise, a disease target), initiates phagocytosis of the target. This triggers two fold action: one, phagocytic engulfment and lysis of the target destroys the target and eliminates it as a first line of immune defense; two, antigens from the target are digested in the phagolysosome of the myeloid cell, are presented on the surface of the myeloid cell, which then leads to activation of T cells and further activation of the immune response and development of immunological memory. Chimeric receptors are engineered for enhanced phagocytosis and immune activation of the myeloid cell in which it is incorporated and expressed. Chimeric antigen receptors of the disclosure are variously termed herein as a chimeric fusion protein, CFP, phagocytic receptor (PR) fusion protein (PFP), or chimeric antigen receptor for phagocytosis (CAR-P), while each term is directed to the concept of a recombinant chimeric and/or fusion receptor protein. In some embodiments, genes encoding non-receptor proteins are also co-expressed in the myeloid cells, typically for an augmentation of the chimeric antigen receptor function. In summary, contemplated herein are various engineered receptor and non-receptor recombinant proteins that are designed to augment phagocytosis and or immune response of a myeloid cell against a disease target, and methods and compositions for creating and incorporating recombinant nucleic acids that encode the engineered receptors or non-receptor recombinant protein, such that the methods and compositions are suitable for creating an engineered myeloid cell for immunotherapy.

In one aspect, the present disclosure provides compositions and methods for stable gene transfer into a cell, where the cell can be any somatic cell. In some embodiments the compositions and methods are designed for cell-specific or tissue-specific delivery. In some cases, the methods described herein relate to supplying a functional protein or a fragment thereof to compensate for an absent or defective (mutated) protein in vivo, e.g., for a protein replacement therapy.

Incorporation of a recombinant nucleic acid in a cell can be accomplished by one or more gene transfer techniques that are available in the state of the art. However, incorporation of exogenous genetic (e.g., nucleic acid) elements into the genome for therapeutic purposes still faces several challenges. Achieving stable integration in a safe and dependable manner, and efficient and prolonged expression are a few among them. Most of the successful gene transfer systems aimed at genomic integration of the cargo nucleic acid sequence rely on viral delivery mechanisms, which have some inherent safety and efficacy issues. Delivery and integration of long nucleic acid sequences cannot be achieved by current gene editing systems.

Little attention has so far been devoted to making and using engineered myeloid cells for stable long-term gene transfer and expression of the transgene. For example, gene transfer to differentiated mammalian cells ex vivo for cell therapy can be accomplished via viral gene transfer mechanisms. However, there are several strategic disadvantages associated with the use of viral gene-transfer vectors, including an undesired potential for transgene silencing over time, the preferential integration into transcriptionally active sites of the genome with associated undesired activation of other genes (e.g. oncogenes) and genotoxicity. In addition to the safety issues increased expense and cumbersome effort of manufacturing, storing and handling integrating viruses often stand in the way of large-scale use of viral vector mediated of gene-modified cells in therapeutic applications. These persistent concerns associated with viral vectors regarding safety, as well as cost and scale of vector production necessitates alternative methods for effective therapy.

Integration of a transgene into the genome of a cell to be used for an immunotherapy can be advantageous in the sense that it is stable and a lower number of cells is required for delivery during the therapy. On the other hand, integrating a transgene in a non-dividing cell can be challenging in both affecting the health and function of the cell as well as the ultimate lifespan of the cell in vivo, and therefore affects its overall utility as the therapeutic. In some embodiments, the methods described herein for generating a myeloid cell for immunotherapy can be a cumulative product of a number of steps and compositions involving but not limited to, for example, selecting a myeloid cell for modifying; method and compositions for incorporating a recombinant nucleic acid in a myeloid cell; methods and compositions for enhancing expression of the recombinant nucleic acid; methods and compositions for selecting and modifying vectors; methods of preparing a recombinant nucleic acid suitable for in vivo administration for uptake and incorporation of the recombinant nucleic acid by a myeloid cell in vivo and therefore generating a myeloid cell for therapy. In some aspects, one or more embodiments of the various inventions described herein are transferrable among each other, and one of skill in the art is expected to use them in alternatives, combinations or interchangeably without the necessity of undue experimentation. All such variations of the disclosed elements are contemplated and fully encompassed herein.

In one aspect, transposons, or transposable elements (TEs) are considered herein, for means of incorporating a heterologous, synthetic or recombinant nucleic acid encoding a transgene of interest in a myeloid cell. Transposon, or transposable elements are genetic elements that have the capability to transpose fragments of genetic material into the genome by use of an enzyme known as transposase. Mammalian genomes contain a high number of transposable element (TE)-derived sequences, and up to 70% of our genome represents TE-derived sequences (de Koning et al. 2011; Richardson et al. 2015). These elements could be exploited to introduce genetic material into the genome of a cell. The TE elements are capable of mobilization, often termed as “jumping” genetic material within the genome. TEs generally exist in eukaryotic genomes in a reversibly inactive, epigenetically silenced form. In the present disclosure methods and compositions for efficient and stable integration of transgenes into macrophages and other phagocytic cells. The method is based on use of a transposase and transposable elements mRNA-encoded transposase. In some embodiments, Long Interspersed Element-1 (L1) RNAs are used for stable integration and/or retrotransposition of the transgene into a cell (e.g., a macrophage or phagocytic cell.

Contemplated herein are methods for retrotransposon mediated stable integration of an exogenous nucleic acid sequence into the genome of a cell. The method may take advantage of the random genomic integration machinery of the retrotransposon into the cell without creating an adverse effect. Methods described herein can be used for robust and versatile incorporation of an exogenous nucleic acid sequence into a cell, such that the exogenous nucleic acid is incorporated at a safe locus within the genome and is expressed without being silenced by the cell's inherent defense mechanism. The method described herein can be used to incorporate an exogenous nucleic acid that is about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb about 8 kb, about 9 kb, about 10 kb, or more in size. In some embodiments, the exogenous nucleic acid is not incorporated within a ribosomal locus. In some embodiments, the exogenous nucleic acid is not incorporated within a ROSA26 locus, or another safe harbor locus. In some embodiments, the methods and compositions described herein can incorporate an exogenous nucleic acid sequence anywhere within the genome of the cell. Furthermore, contemplated herein is a retrotransposition system that is developed to incorporate an exogenous nucleic acid sequence into a specific predetermined site within the genome of a cell, without creating an adverse effect. The disclosed methods and compositions incorporate several mechanisms of engineering the retrotransposons for highly specific incorporation of the exogenous nucleic acid into a cell with high fidelity. Retrotransposons chosen for this purpose may be a human retrotransposon.

Methods and compositions described herein represent a salient breakthrough in the molecular systems and mechanisms for manipulating the genome of a cell. Shown here for the first time is a method that exploits a human retrotransposon system into non-virally delivering and stably integrating a large fragment of exogenous nucleic acid sequence (at least greater than 100 nucleobases, at least greater than 1 kb, at least greater than 2 kb, at least greater than 3 kb, etc.) into a non-conserved region of the genome that is not an rDNA or a ribosomal locus or a designated safe-harbor locus such as the ROSA 26 locus.

In some embodiments, a retrotransposable system is used to stably incorporate into the genome and express a non-endogenous nucleic acid, where the non-endogenous nucleic acid comprises retrotransposable elements within the nucleic acid sequence. In some embodiments, a cell's endogenous retrotransposable system (e.g., proteins and enzymes) is used to stably express a non-endogenous nucleic acid in the cell. In some embodiments, a cell's endogenous retrotransposable system (e.g., proteins and enzymes, such as a LINE-1 retrotransposition system) is used, but may further express one or more components of the retrotransposable system to stably express a non-endogenous nucleic acid in the cell.

In some embodiments, a synthetic nucleic acid is provided herein, the synthetic nucleic acid encoding a transgene, and encoding one or more components for genomic integration and/or retrotransposition.

In one aspect, provided herein is a method of integrating a nucleic acid sequence into a genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA into the cell, wherein the mRNA comprises: an insert sequence, wherein the insert sequence comprises an exogenous sequence, or a sequence that is a reverse complement of the exogenous sequence; a 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for a human ORF protein, and wherein the insert sequence is integrated into the genome of the cell. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for human ORF2p.

In one aspect, provided herein is a method for integrating a nucleic acid sequence into the genome of an immune cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence, wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and wherein the transgene sequence is integrated into the genome of the immune cell.

In one aspect, provided herein is a method for integrating a nucleic acid sequence into the genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; a 5′ UTR sequence, a sequence of a human retrotransposon downstream of the 5′ UTR sequence, and a 3′ UTR sequence downstream of the sequence of a human retrotransposon; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and wherein the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs, and wherein the insert sequence is integrated into the genome of the cell.

In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises an ORF2p binding site. In some embodiments, the ORF2p binding site is a poly A sequence in the 3′ UTR sequence.

In some embodiments, the mRNA comprises a sequence of a human retrotransposon. In some embodiments, the sequence of a human retrotransposon is downstream of the 5′ UTR sequence. In some embodiments, the sequence of a human retrotransposon is upstream of the 3′ UTR sequence. In some embodiments, the polynucleotide sequence that is desired to be transferred and incorporated into the genome of a cell (e.g., the insert) is inserted at a site 3′ to the sequence encoding ORF1 in a recombinant nucleic acid construct. In some embodiments, the polynucleotide sequence that is desired to be transferred and incorporated into the genome of a cell is inserted at a site 3′ to the sequence encoding ORF2 in a recombinant nucleic acid construct. In some embodiments the sequence that is desired to be transferred and incorporated into the genome of a cell is inserted within the 3′-UTR of ORF1 or ORF2, or both. In some embodiments, the polynucleotide sequence that is sequence that is desired to be transferred and incorporated into the genome of a cell is inserted upstream of the poly A tail of ORF2 in a recombinant nucleic acid construct.

In some embodiments, the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs. In some embodiments, the two ORFs are non-overlapping ORFs. In some embodiments, the two ORFs are ORF1 and ORF2. In some embodiments, the ORF1 encodes ORF1p and ORF2 encodes ORF2p.

In some embodiments, the sequence of a human retrotransposon comprises a sequence of a non-LTR retrotransposon. In some embodiments, the sequence of a human retrotransposon comprises a LINE-1 retrotransposon. In some embodiments, the LINE-1 retrotransposon is a human LINE-1 retrotransposon. In some embodiments, the sequence of a human retrotransposon comprises a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the endonuclease and/or a reverse transcriptase is ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase domain. In some embodiments, the endonuclease and/or a reverse transcriptase is a minke whale endonuclease and/or a reverse transcriptase. In some embodiments, the sequence of a human retrotransposon comprises a sequence encoding ORF2p. In some embodiments, the insert sequence is integrated into the genome at a poly T site using specificity of an endonuclease domain of the ORF2p. In some embodiments, the poly T site comprises the sequence TTTTTA.

In some embodiments, provided herein is a polynucleotide construct comprising an mRNA wherein the mRNA comprises a sequence encoding a human retrotransposon, wherein, (i) the sequence of a human retrotransposon comprises a sequence encoding ORF1p, (ii) the mRNA does not comprise a sequence encoding ORF1p, or (iii) the mRNA comprises a replacement of the sequence encoding ORF1p with a 5′ UTR sequence from the complement gene. In some embodiments, the mRNA comprises a first mRNA molecule encoding ORF1p, and a second mRNA molecule encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the mRNA is an mRNA molecule comprising a first sequence encoding ORF1p, and a second sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase are separated by a linker sequence.

In some embodiments, the linker sequence comprises an internal ribosome entry sequence (IRES). In some embodiments, the IRES is an IRES from CVB3 or EV71. In some embodiments, the linker sequence encodes a self-cleaving peptide sequence. In some embodiments, the linker sequence encodes a T2A, a E2A or a P2A sequence

In some embodiments, the sequence of a human retrotransposon comprises a sequence that encodes ORF1p fused to an additional protein sequence and/or a sequence that encodes ORF2p fused to an additional protein sequence. In some embodiments, the ORF1p and/or the ORF2p is fused to a nuclear retention sequence. In some embodiments, the nuclear retention sequence is an Alu sequence. In some embodiments, the ORF1p and/or the ORF2p is fused to an MS2 coat protein. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises at least one, two, three or more MS2 hairpin sequences. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that promotes or enhances interaction of a poly A tail of the mRNA with the endonuclease and/or a reverse transcriptase. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that promotes or enhances interaction of a poly-A-binding proteins (e.g., PABP) with the endonuclease and/or a reverse transcriptase. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that increases specificity of the endonuclease and/or a reverse transcriptase to the mRNA relative to another mRNA expressed by the cell. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises an Alu element sequence.

In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase have the same promoter. In some embodiments, the insert sequence has a promoter that is different from the promoter of the first sequence encoding ORF1p. In some embodiments, the insert sequence has a promoter that is different from the promoter of the second sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and/or the second sequence encoding an endonuclease and/or a reverse transcriptase have a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site and combinations thereof. In some embodiments, the insert sequence has a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site and combinations thereof.

In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase are codon optimized for expression in a human cell.

In some embodiments, the mRNA comprises a WPRE element. In some embodiments, the mRNA comprises a selection marker. In some embodiments, the mRNA comprises a sequence encoding an affinity tag. In some embodiments, the affinity tag is linked to the sequence encoding an endonuclease and/or a reverse transcriptase.

In some embodiments, the 3′ UTR comprises a poly A sequence or wherein a poly A sequence is added to the mRNA in vitro. In some embodiments, the poly A sequence is downstream of a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the insert sequence is upstream of the poly A sequence.

In some embodiments, the 3′ UTR sequence comprises the insert sequence. In some embodiments, the insert sequence comprises a sequence that is a reverse complement of the sequence encoding the exogenous polypeptide. In some embodiments, the insert sequence comprises a polyadenylation site. In some embodiments, the insert sequence comprises an SV40 polyadenylation site. In some embodiments, the insert sequence comprises a polyadenylation site upstream of the sequence that is a reverse complement of the sequence encoding the exogenous polypeptide. In some embodiments, the insert sequence is integrated into the genome at a locus that is not a ribosomal locus. In some embodiments, the insert sequence is integrated into the genome at a locus that is not a rDNA locus. In some embodiments, the insert sequence integrates into a gene or regulatory region of a gene, thereby disrupting the gene or downregulating expression of the gene. In some embodiments, the insert sequence integrates into a gene or regulatory region of a gene, thereby upregulating expression of the gene. In some embodiments, the insert sequence integrates into the genome and replaces a gene. In some embodiments, the insert sequence is stably integrated into the genome. In some embodiments, the insert sequence is retrotransposed into the genome. In some embodiments, the insert sequence is integrated into the genome by cleavage of a DNA strand of a target site by an endonuclease encoded by the mRNA. In some embodiments, the insert sequence is integrated into the genome via target-primed reverse transcription (TPRT). In some embodiments, the insert sequence is integrated into the genome via reverse splicing of the mRNA into a DNA target site of the genome.

In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the immune cell is a myeloid cell. In some embodiments, the immune cell is selected from a group consisting of a monocyte, a macrophage, a dendritic cell, a dendritic precursor cell, and a macrophage precursor cell.

In some embodiments, the mRNA is a self-integrating mRNA. In some embodiments, the method comprises introducing into the cell the mRNA. In some embodiments, the method comprises introducing into the cell the vector encoding the mRNA. In some embodiments, the method comprises introducing the mRNA or the vector encoding the mRNA into a cell ex vivo. In some embodiments, the method further comprises administering the cell to a human subject. In some embodiments, the method comprises administering the mRNA or the vector encoding the mRNA to a human subject. In some embodiments, an immune response is not elicited in the human subject. In some embodiments, the mRNA or the vector is substantially non-immunogenic.

In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector comprises a non-LTR retrotransposon. In some embodiments, the vector comprises a human L1 element. In some embodiments, the vector comprises a L1 retrotransposon ORF1 gene. In some embodiments, the vector comprises a L1 retrotransposon ORF2 gene. In some embodiments, the vector comprises a L1 retrotransposon. In some embodiments, provided herein is an mRNA comprising sequences encoding human LINE 1 retrotransposition elements, and a payload comprising a nucleic acid sequence which can be retrotransposed and integrated into a genome of a cell comprising the mRNA. In some embodiments, provided herein is an mRNA that can be delivered into a living cell, e.g., a human cell, wherein, the mRNA comprises sequences encoding human LINE 1 retrotransposition elements, and a payload comprising a nucleic acid sequence which can be retrotransposed and integrated into the genome of the cell. In some embodiments, the sequences encoding human LINE 1 retrotransposition elements comprise a L1 retrotransposon ORF1 sequence or a fragment thereof. In some embodiments, the sequences encoding human LINE 1 retrotransposition elements comprise a L1 retrotransposon ORF2 sequence or a fragment thereof. In some embodiments, the sequences encoding human LINE 1 retrotransposition elements comprise a L1 retrotransposon ORF1 sequence or a fragment thereof and a L1 retrotransposon ORF2 sequence or a fragment thereof, and a nucleic acid “payload” sequence which is a heterologous sequence which is integrated into the genome of cell by retrotransposition. (See, for example, FIG. 1B).

In some embodiments, the mRNA is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases. In some embodiments, the mRNA is a most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 kilobases. In some embodiments, the mRNA is at least about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6 kilobases. In some embodiments, the mRNA is at least about 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7 kilobases. In some embodiments, the mRNA is at least about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 kilobases. In some embodiments, the mRNA is at least about 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9 kilobases. In some embodiments, the mRNA is at least about 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 kilobases.

In some embodiments, the mRNA comprises a sequence that inhibits or prevents degradation of the mRNA. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA inhibits or prevents degradation of the mRNA by an exonuclease or an RNAse. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA is a G quadruplex, pseudoknot or triplex sequence. In some embodiments, the sequence the sequence that inhibits or prevents degradation of the mRNA is an exoribonuclease-resistant RNA structure from a flaviviral RNA or an ENE element from KSV. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA inhibits or prevents degradation of the mRNA by a deadenylase. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA comprises non-adenosine nucleotides within or at a terminus of a poly A tail of the mRNA. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA increases stability of the mRNA. In some embodiments, the exogenous sequence comprises a sequence encoding an exogenous polypeptide. In some embodiments, the sequence encoding an exogenous polypeptide is not in frame with a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the sequence encoding an exogenous polypeptide is not in frame with a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the exogenous sequence does not comprise introns. In some embodiments, the exogenous sequence comprises a sequence encoding an exogenous polypeptide selected from the group consisting of an enzyme, a receptor, a transport protein, a structural protein, a hormone, an antibody, a contractile protein and a storage protein. In some embodiments, the exogenous sequence comprises a sequence encoding an exogenous polypeptide selected from the group consisting of a chimeric antigen receptor (CAR), a ligand, an antibody, a receptor, and an enzyme. In some embodiments, the exogenous sequence comprises a regulatory sequence. In some embodiments, the regulatory sequence comprises a cis-acting regulatory sequence. In some embodiments, the regulatory sequence comprises a cis-acting regulatory sequence selected from the group consisting of an enhancer, a silencer, a promoter or a response element. In some embodiments, the regulatory sequence comprises a trans-acting regulatory sequence. In some embodiments, the regulatory sequence comprises a trans-acting regulatory sequence that encodes a transcription factor.

In some embodiments, integration of the insert sequence does not adversely affect cell health. In some embodiments, the endonuclease, the reverse transcriptase or both are capable of site-specific integration of the insert sequence.

In some embodiments, the mRNA comprises a sequence encoding an additional nuclease domain or a nuclease domain that is not derived from ORF2. In some embodiments, the mRNA comprises a sequence encoding a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a zinc finger binding domain from an R2 retroelement, or a DNA binding domain that binds to repetitive sequences such as a Rep78 from AAV. In some embodiments, the endonuclease comprises a mutation that reduces activity of the endonuclease compared to the endonuclease without the mutation. In some embodiments, the endonuclease is an ORF2p endonuclease and the mutation is S228P. In some embodiments, the mRNA comprises a sequence encoding a domain that increases fidelity and/or processivity of the reverse transcriptase. In some embodiments, the reverse transcriptase is a reverse transcriptase from a retroelement other than ORF2 or reverse transcriptase that has higher fidelity and/or processivity compared to a reverse transcriptase of ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is a group IIA intron reverse transcriptase, a group IIB intron reverse transcriptase, or a group IIC intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is TGIRT-II or TGIRT-III.

In some embodiments, the mRNA comprises a sequence comprising an Alu element and/or a ribosome binding aptamer. In some embodiments, the mRNA comprises a sequence encoding a polypeptide comprising a DNA binding domain. In some embodiments, the 3′ UTR sequence is derived from a viral 3′ UTR or a beta-globin 3′ UTR.

In one aspect, provided herein is a composition comprising a recombinant mRNA or vector encoding an mRNA, wherein the mRNA comprises a human LINE-1 transposon sequence comprising a human LINE-1 transposon 5′ UTR sequence, a sequence encoding ORF1p downstream of the human LINE-1 transposon 5′ UTR sequence, an inter-ORF linker sequence downstream of the sequence encoding ORF1p, a sequence encoding ORF2p downstream of the inter-ORF linker sequence, and a 3′ UTR sequence derived from a human LINE-1 transposon downstream of the sequence encoding ORF2p; wherein the 3′ UTR sequence comprises an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element.

In some embodiments, the insert sequence integrates into the genome of a cell when introduced into the cell. In some embodiments, the insert sequence integrates into a gene associated a condition or disease, thereby disrupting the gene or downregulating expression of the gene. In some embodiments, the insert sequence integrates into a gene, thereby upregulating expression of the gene. In some embodiments, the recombinant mRNA or vector encoding the mRNA is isolated or purified.

In one aspect, provided herein is a composition comprising a nucleic acid comprising a nucleotide sequence encoding (a) a long interspersed nuclear element (LINE) polypeptide, wherein the LINE polypeptide includes human ORF1p and human ORF2p; and (b) an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element, wherein the composition is substantially non-immunogenic.

In some embodiments, the composition comprises human ORF1p and human ORF2p proteins. In some embodiments, the composition comprises a ribonucleoprotein (RNP) comprising human ORF1p and human ORF2p complexed to the nucleic acid. In some embodiments, the nucleic acid is mRNA.

In one aspect, provided herein is a composition comprising a cell comprising a composition described herein. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the immune cell is a myeloid cell. In some embodiments, the immune cell is selected from a group consisting of a monocyte, a macrophage, a dendritic cell, a dendritic precursor cell, and a macrophage precursor cell. In some embodiments, the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide and the exogenous polypeptide is a chimeric antigen receptor (CAR).

In one aspect, provided herein is a pharmaceutical composition comprising a composition described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is for use in gene therapy. In some embodiments, the pharmaceutical composition is for use in the manufacture of a medicament for treating a disease or condition. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition. In one aspect, provided herein is a method of treating a disease in a subject, comprising administering a pharmaceutical composition described herein to a subject with a disease or condition. In some embodiments, the method increases an amount or activity of a protein or functional RNA in the subject. In some embodiments, the subject has a deficient amount or activity of a protein or functional RNA. In some embodiments, the deficient amount or activity of a protein or functional RNA is associated with or causes the disease or condition.

In some embodiments, the method further comprising administering an agent that inhibits human silencing hub (HUSH) complex, an agent that inhibits FAM208A, or an agent that inhibits TRIM28. In some embodiments, the agent that inhibits human silencing hub (HUSH) complex is an agent that inhibits Periphilin, TASOR and/or MPP8. In some embodiments, the agent that inhibits human silencing hub (HUSH) complex inhibits assembly of the HUSH complex. In some embodiments, the agent inhibits the fanconia anemia complex. In some embodiments, the agent inhibits FANCD2-FANC1 heterodimer monoubiquitination. In some embodiments, the agent inhibits FANCD2-FANC1 heterodimer formation. In some embodiments the agent inhibits the Fanconi Anemia (FA) core complex. FA core complex is a component of the fanconi anemia DNA damage repair pathway, e.g., in chemotherapy induced DNA inter-strand crosslinks. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Examples of agents that can inhibit the FA core complex include Bortezomib and curcumin analogs EF24 and 4H-TTD.

Accordingly, it is an object of the present invention to provide novel transposon-based vectors useful in providing gene therapy to an animal. It is an object of the present invention to provide novel transposon-based vectors for use in the preparation of a medicament useful in providing gene therapy to an animal or human. It is another object of the present invention to provide novel transposon-based vectors that encode for the production of desired proteins or peptides in cells. Yet another object of the present invention to provide novel transposon-based vectors that encode for the production of desired nucleic acids in cells. It is a further object of the present invention to provide methods for cell and tissue specific incorporation of transposon-based DNA or RNA constructs comprising targeting a selected gene to a specific cell or tissue of an animal. It is yet another object of the present invention to provide methods for cell and tissue specific expression of transposon-based DNA or RNA constructs comprising designing a DNA or RNA construct with cell specific promoters that enhance stable incorporation of the selected gene by the transposase and expressing the selected gene in the cell. It is an object of the present invention to provide gene therapy for generations through germ line administration of a transposon-based vector. Another object of the present invention is to provide gene therapy in animals through non germ line administration of a transposon-based vector. Another object of the present invention is to provide gene therapy in animals through administration of a transposon-based vector, wherein the animals produce desired proteins, peptides or nucleic acids. Yet another object of the present invention is to provide gene therapy in animals through administration of a transposon-based vector, wherein the animals produce desired proteins or peptides that are recognized by receptors on target cells. Still another object of the present invention is to provide gene therapy in animals through administration of a transposon-based vector, wherein the animals produce desired fusion proteins or fusion peptides, a portion of which are recognized by receptors on target cells, in order to deliver the other protein or peptide component of the fusion protein or fusion peptide to the cell to induce a biological response. Yet another object of the present invention is to provide a method for gene therapy of animals through administration of transposon-based vectors comprising tissue specific promoters and a gene of interest to facilitate tissue specific incorporation and expression of a gene of interest to produce a desired protein, peptide or nucleic acid. Another object of the present invention is to provide a method for gene therapy of animals through administration of transposon-based vectors comprising cell specific promoters and a gene of interest to facilitate cell specific incorporation and expression of a gene of interest to produce a desired protein, peptide or nucleic acid. Still another object of the present invention is to provide a method for gene therapy of animals through administration of transposon-based vectors comprising cell specific promoters and a gene of interest to facilitate cell specific incorporation and expression of a gene of interest to produce a desired protein, peptide or nucleic acid, wherein the desired protein, peptide or nucleic acid has a desired biological effect in the animal.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “FIG.” herein), of which:

FIG. 1A illustrates a general mechanism of action of retrotransposons. (I) is a schematic representing the overall lifecycle of an autonomous retrotransposon. (II) LINE-1 retrotransposon comprises LINE-1 elements, which encode two proteins ORF1p and ORF2p that are expressed as mRNAs. The bicistronic mRNA is translated into the two proteins, and when ORF2p is translated by a read-through event by the ribosome, it binds the 3′ end of its own mRNA through the poly A tail (III). ORF2p cleaves at a consensus sequence TAAAA, where the poly A at the 3′ end of the mRNA hybridizes and primes the reverse transcriptase activity of the ORF2 protein. The protein reverse-transcribes the mRNA back into DNA leading to an insertion of the LINE-1 sequence back into a new location in the genome (IV).

FIG. 1B is an illustration of a schematic diagram of an mRNA construct that comprises a genetic payload (left) that can be designed for integration into the genome (right).

FIG. 1C illustrates various exemplary designs for integrating an mRNA encoding a transgene into the genome of a cell. GFP shown here in a box is an exemplary transgene.

FIG. 1D illustrates various exemplary designs for integrating an mRNA encoding a transgene into the genome of a cell. GFP shown here in a box is an exemplary transgene.

FIG. 1E is an illustration a schematic of the LINE-1 retrotransposition cycle showing the mechanism of action of the LINE transposons and introduction of a transgene cargo into a retrotransposon cite. LINE-1 retrotransposons are genomic sequences that encode for two proteins, ORF1 and ORF2. These elements are transcribed and translated into proteins that form an RNA-protein complex with the LINE-1 mRNA, ORF1 trimers, and ORF2, a reverse-transcriptase endonuclease. This complex translocates back into the nuclease where it cleaves DNA at a 5′-TTTT N-3′ motif and is primed for reverse-transcription of the LINE-1 RNA by the ORF2 protein by making an RNA-DNA hybrid with the poly A tail of the mRNA and the resected cleaved DNA. Reverse-transcription of the LINE-1 into cDNA leads to a new LINE-1 integration event.

FIG. 2A illustrates three exemplary designs for expressing an exemplary transgene GFP by stably incorporating the sequence encoding GFP using the constructs. Expected GFP expression levels at 72 hours are shown on the right side.

FIG. 2B illustrates three exemplary designs for expressing an exemplary transgene GFP by stably incorporating the sequence encoding RFP, RFP and GFP or ORF2p and GFP using the constructs. Expected GFP and RFP expression levels at 72 hours are shown on the right side.

FIG. 3A illustrates an exemplary diagram of conventional circRNA structure and formation.

FIG. 3B illustrates two views of an exemplary RL-GAAA tectoRNA motif designs.

FIG. 3C illustrates exemplary structures of chip-flow piece RNAs as platforms for testing potential tectoRNA.

FIG. 4A illustrates an exemplary schematic showing ORF2p binding to an ORF2 poly A region.

FIG. 4B illustrates an exemplary schematic showing how a fusion of ORF2p with an MS2 RNA binding domain binds to an MS2 binding RNA sequence in the 3′UTR of an mRNA encoding the ORF2 an increase specificity.

FIG. 4C illustrates exemplary designs of retrotransposon systems for stably integrating a nucleic acid into the genome of a cell at specific sites. The upper panel shows a design using an ORFp2-MegaTAL DNA binding domain fusion where the DNA binding and endonuclease activity of ORF2p is mutated to be inactive. The middle panel shows a chimeric ORF2p where the endonuclease domain has been replaced with a high specificity and high-fidelity nuclease domain of another protein. The lower panel shows a fusion of a DNA binding domain of a heterologous protein with ORF2p such that the fusion protein binds to ORF2 binding site as well additional DNA sequences in the vicinity of the ORF2 site.

FIG. 5 illustrates exemplary constructs (I)-(X) for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 6A illustrates an exemplary construct with a sequence encoding ORF1p for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 6B illustrates an exemplary construct without a sequence encoding ORF1p for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 7A illustrates exemplary methods of improving mRNA half-life by inhibiting degradation by 5′-3′ exonucleases, such as XRN1, or 3′-5′ exosomal degradation, by introducing structures corresponding to a G-quadruplex, or, a pseudoknot (SEQ ID NO: 82) in the 5′UTR; and/or xrRNAs, a triplex motifs (SEQ ID NOS. 83-85 in order of appearance) and/or a non-A nucleotide residues in the 3′UTR.

FIG. 7B illustrates an exemplary schematic of a myeloid cell expressing a transgene encoding a chimeric receptor that binds a cancer cell and induces anti-cancer activity.

FIG. 7C shows expected results of introducing bulk or purified RNA encoding a chimeric receptor that binds a cancer cell as described in FIG. 7B on increased and prolonged expression of the chimeric receptors.

FIG. 8A shows an exemplary plasmid design and expected LINE-1 mRNA transcript with a cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (comprising ORF1 and ORF2 protein encoding sequences) and a cargo sequence which is a nucleic acid sequence encoding GFP, where the coding sequence of GFP is interrupted with an intron. The GFP is not expressed until the sequence is integrated in the genome and the intron is spliced.

FIG. 8B shows exemplary results showing successful integration of the mRNA transcript encoded by the plasmid shown in FIG. 8A and expression of GFP relative to mock-transfected cells (fold increase in mean fluorescence intensity of GFP positive cells is shown). Mock transfected cells were transfected by the vector lacking the GFP cargo sequence.

FIG. 8C shows exemplary flow cytometry results from the results shown in FIG. 8B.

FIG. 9A shows an exemplary plasmid design and expected LINE-1 mRNA transcript with a cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (comprising ORF1 and ORF2 protein encoding sequences) and a cargo sequence which is a nucleic acid sequence encoding a recombinant chimeric fusion receptor protein (ATAK receptor) that has extracellular region capable of binding to CD5 and an intracellular region comprising an FCR intracellular domain and a PI3 kinase recruitment domain. The coding sequence of the ATAK receptor is interrupted with an intron.

FIG. 9B shows exemplary results showing successful integration of the mRNA transcript encoded by the plasmid shown in FIG. 9A and expression of ATAK relative to mock-transfected cells (fold increase in mean fluorescence intensity of ATAK positive cells is shown). Mock transfected cells were transfected by the vector lacking the ATAK cargo sequence. Expression of ATAK receptor protein was detected by binding with a labeled CD5 antibody.

FIG. 9C shows exemplary flow cytometry results from the results shown in FIG. 9B.

FIG. 10A shows an exemplary plasmid design and expected LINE-1 mRNA transcript with a cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (comprising ORF1 and ORF2 protein encoding sequences) and a cargo sequence which is a nucleic acid sequence encoding a recombinant chimeric fusion receptor protein (ATAK receptor) followed by a T2A self-cleavage sequence followed by a split GFP sequence (all in a reverse orientation relative to the LINE-1 sequence). The coding sequence of the GFP is interrupted with an intron. Expected mRNA after reverse transcription and integration of the cargo are depicted.

FIG. 10B shows exemplary results showing successful integration of the mRNA transcript encoded by the plasmid shown in FIG. 10A and expression of ATAK-T2A-GFP relative to mock-transfected cells (fold change in GFP and ATAK double positive cells is shown). Mock transfected cells were transfected by the vector lacking the ATAK cargo sequence. Expression of ATAK receptor protein was detected by binding with a labeled CD5 antibody.

FIG. 10C shows representative flow cytometry data from two separate experimental runs for expression of both GFP and CD5 binder (ATAK) using the experimental setup shown in FIG. 10A.

FIG. 10D shows representative flow cytometry data from two separate experimental runs for expression of both GFP and CD5 binder (ATAK) using the experimental setup shown in FIG. 10A.

FIG. 11A shows exemplary mRNA constructs for retrotransposition-based gene delivery. The ORF1 and ORF2 sequences are in two difference mRNA molecules. The ORF2p (ORF2) coding mRNA comprises and inverted GFP coding sequence.

FIG. 11B depicts exemplary data showing expression of GFP (fold increase in mean fluorescence intensity of GFP positive cells is shown) upon electroporating both ORF1-mRNA and ORF2-FLAG-GFPai mRNA normalized to electroporation of ORF2-FLAG-GFPai mRNA only.

FIG. 12A depicts exemplary data showing expression of GFP (fold increase in mean fluorescence intensity of GFP positive cells is shown) upon electroporating ORF1-mRNA and ORF2-FLAG-GFPai mRNA at different amounts. Fold increase is relative to 1×ORF2-GFPao and 1×ORF1 mRNA.

FIG. 12B shows an exemplary fluorescent microscopy image of GFP+ cells following electroporation of the mRNA depicted in FIG. 11A.

FIG. 13A shows exemplary mRNA constructs where the ORF1 and ORF2 sequences are in two difference mRNA molecules (top panel) and a LINE-1 mRNA transcript comprising ORF1 and ORF2 protein encoding sequences on a single mRNA molecule (bottom panel) for gene delivery. mRNA contains the bicistronic ORF1 and ORF2 sequence with a CMV-GFP sequence in the 3′UTR going from 3′-5′. Upon retrotransposition of the delivered ORF2-cmv-GFP antisense (LINE-1 mRNA), cells are expected to express GFP.

FIG. 13B depicts exemplary data showing expression of GFP (fold increase in mean fluorescence intensity of GFP positive cells is shown) upon electroporating the constructs depicted in FIG. 13A.

FIG. 14A shows an exemplary experimental design for testing whether multiple electroporations increases retrotransposition efficiency. HEK293T cells were electroporated every 48 hours with the Maxcyte system and assessed for GFP positive cells using flow after culturing for 24-72 hrs.

FIG. 14B depicts exemplary data showing expression of GFP at the indicated times (fold increase in mean fluorescence intensity of GFP positive cells is shown) upon electroporating 1-5 times according to FIG. 14A.

FIG. 15A depicts exemplary constructs to enhance retrotransposition via mRNA delivery. In one construct a nuclear localization signal (NLS) sequence is fused to the C terminus of the ORF2 sequence (ORF2-NLS fusion). In one construct a Minke whale ORF2 sequence was used in place of the human ORF2. In one construct a minimal sequence of the Alu element (AJL-H33delta) is inserted in the 3′UTR of the LINE-1 sequence. In one construct MS2 hairpins are inserted in the 3′UTR of the LINE-1 sequence and an MS2 hairpin binding protein (MCP) sequence is fused to the ORF2 sequence.

FIG. 15B depicts exemplary data showing expression of GFP (fold increase in mean fluorescence intensity of GFP positive cells is shown) using the constructs depicted in FIG. 15A.

FIG. 16A shows exemplary plasmid constructs where the ORF1 and ORF2 sequences are in two difference plasmid molecules (top panel) and a plasmid encoding a LINE-1 mRNA transcript comprising ORF1 and ORF2 protein encoding sequences on a single mRNA molecule with various replacements of the inter-ORF sequence between ORF1 and ORF2 (bottom panel) for gene delivery.

FIG. 16B depicts exemplary data showing expression of GFP (fold increase in mean fluorescence intensity of GFP positive cells is shown) using the constructs depicted in FIG. 16A.

FIG. 17A depicts an exemplary plasmid construct encoding a LINE-1 mRNA transcript comprising ORF1 and ORF2 protein encoding sequences on a single mRNA molecule with a GFP sequence (top panel) and an exemplary LINE-1 mRNA transcript comprising ORF1 and ORF2 protein encoding sequences on a single mRNA molecule with a GFP sequence.

FIG. 17B depicts exemplary data showing expression of GFP (fold increase in mean fluorescence intensity of GFP positive cells is shown) in Jurkat cells using the constructs depicted in FIG. 17A. The plasmid construct was transfected, and the mRNA construct was electroporated.

FIG. 18A shows an exemplary plasmid design and expected LINE-1 mRNA transcript with a cargo nucleic acid sequence. The plasmid has a LINE-1 sequence (comprising ORF1 and ORF2 protein encoding sequences) and a cargo sequence which is a nucleic acid sequence encoding a recombinant chimeric fusion receptor protein (ATAK receptor) followed by a T2A self-cleavage sequence followed by a split GFP sequence (all in a reverse orientation relative to the LINE-1 sequence). The coding sequence of the GFP is interrupted with an intron. Expected mRNA after reverse transcription and integration of the cargo are depicted.

FIG. 18B shows exemplary results showing successful integration of the mRNA transcript encoded by the plasmid shown in FIG. 10A and expression of ATAK-T2A-GFP relative to mock-transfected cells (fold change in GFP and ATAK double positive cells is shown) in a myeloid cell line (THP-1). Data represents expression at 6 days post transfection, normalized over mock plasmid transfected cells wherein the mock plasmid does not have GFP coding sequence.

FIG. 19 illustrates an exemplary experimental set up for cell synchronization. A heterogenous cell population is sorted based on cell cycle stage, prior to delivery of an exogenous nucleic acid. Cell cycle synchronization is expected to result in higher expression and stabilization of the exogenous nucleic acid delivered. If cells are not homogeneous after cell sorting, then cells can be further incubated with a suitable agent that arrests cell cycle at a stage.

FIG. 20 illustrates an exemplary method for increasing retrotransposon efficiency by inducing DNA double stranded breaks, with or without inhibiting DNA repair pathways, such as by inducing DNA ligase inhibitor SCR7 or inhibiting host surveillance proteins, for example, using miRNA to HUSH complex TASOR protein.

FIG. 21 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 22 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 23 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 24 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 25 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 26 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 27 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 28 illustrates exemplary constructs for integrating an mRNA encoding a transgene into the genome of a cell.

FIG. 29 illustrates exemplary retrotransposon constructs (left) with a 2.4 kb cargo with a general mechanism of action of the retrotransposon, and a representative data (right) for expression of a fluorescent GFP marker encoded by the cargo from a nucleic acid sequence integrated into the genome in HEK293 cells. Placement of an antisense GFP gene split with an intron in the sense direction and a promoter sequence in the 3′UTR of the LINE-1 leads to reconstitution and retrotransposition of the GFP cargo. GFP expression in 293T cells transfected with the construct shown on the left, as measured by flow cytometry (right) and quantitated bar graphs (bottom left). Data collected 35 days after doxycycline induction of the ORF.

FIG. 30 illustrates exemplary retrotransposon constructs (left) with a 3.0 kb cargo comprising a membrane protein (CD5 binder chimeric antigen receptor, CD5-CAR), and a representative flow cytometry data for expression of the CD5 binder (right) from the nucleic acid sequence integrated into the genome in HEK293 cells. % of CD5 binder positive (+) cells is indicated in the inset.

FIG. 31 illustrates an exemplary retrotransposon construct (top) with a 3.7 kb cargo comprising a membrane protein (CD5 binder chimeric antigen receptor, CD5-CAR and a GFP separated by an auto-cleavable T2A element), and a representative flow cytometry data (bottom) demonstrating the expression of the CD5 binder and GFP.

FIG. 32 illustrates an exemplary retrotransposon construct (top) with a 3.9 kb cargo comprising a membrane protein (HER2 binder chimeric antigen receptor, and a GFP separated by an auto-cleavable T2A element), and a representative flow cytometry data (bottom) demonstrating the expression of the HER2 binder and GFP.

FIG. 33A shows exemplary data for delivery of retrotransposon elements delivered as mRNA.

FIG. 33B shows schematic diagram showing a trans and a cis mRNA design for delivery of LINE 1 mRNA with GFP cargo (top panel). Representative results of electroporation of 293T cells with trans mRNAs with separate ORF1 and ORF2 mRNAs. 293T cells were electroporated with 100 ug/mL of mRNA either with ORF2 alone, ORF1+ORF2 mRNAs, each at 100 ug/mL, or a GFP-encoding mRNA with the same 5′ and 3′UTRs as the ORF1 mRNA (left panel of data plots). Retrotransposition events result in GFP-positive cells. Cells were assayed for GFP fluorescence by flow cytometry 4 days and 10 days post-electroporation. Mock electroporated cells serve as the negative control population for gating. Bar graph on the right shows results from a representative experiment indicating titration of trans mRNAs and cis ORF1 and ORF2 containing mRNA concentration during electroporation. Trans mRNAs solid bars and cis mRNA stripes. 20× is 2000 ug/mL in the electroporation reaction.

FIG. 33C shows titration of the ORF1 and ORF2-GFPai trans mRNAs. Increasing the concentration separately and together during the electroporation to 200 ug/mL increases retrotransposition of the GFP gene cargo.

FIG. 33D illustrates an exemplary data for the different constructs indicated above each flow cytometry data plot in the figure, the top panel on day 4, and the bottom panel on day 13. Right hand figures illustrate light and fluorescent microscopic images of a the GFP expressing cells in culture. Copies of integrated cargo per construct is demonstrated in the bottom right at day 13. qPCR assay for genomic DNA integration from different LINE-1 plasmid transfected, LINE-1 mRNA (retro-mRNA), and ORF1 and ORF2-GFP mRNA electroporated cells is shown. Two qPCR primer-probe sets were used, one for the housekeeping gene RPS30 and the other for the GFP gene. Plasmid-transfected cells use a plasmid that does not contain and SV40 maintenance sequence. Integration per cell is calculated from determining copy numbers per samples through interpolation of a standard curve of plasmid and genomic DNA, and normalizing for the two copies of RPS30 per 293T cell. Error bard denote standard deviation of three technical replicate measurements.

FIG. 34 illustrates exemplary retrotransposon construct (left) and expression data (right) in the indicated cell lines.

FIG. 35 illustrates flow cytometry data showing expression of LINE 1 GFP constructs in K562, 293T and THP1 cells (upper panel); and number of integrations of LINE-2-GFP mRNA per cell in K562 and THP-1 cell lines (lower panel).

FIG. 36 illustrates flow cytometry data showing expression of LINE 1 GFP constructs in primary T cells (left). Integrations per cell are indicated in the graph on the right. Data was collected on day 6 after electroporation.

FIG. 37A shows a schematic of activation, culture times, electroporation, and GFP expression assay of isolated primary T cells.

FIG. 37B illustrates flow cytometry data showing expression of LINE 1 GFP mRNA constructs in primary T cells at the indicated concentrations and before and after freeze-thaw as indicated in the figure. Integrations per cell is shown in the bar diagram. GFP expression using a retro-mRNA electroporation with a GFP cargo. GFP expression was assayed 4 days post electroporation and 15 days of culturing post electroporation. Primary T cells were cryo-preserved and thawed during this time. qPCR integration assay for GFP integration. Genomic DNA from the 20× sample was isolated and assayed for copies of GFP.

FIG. 38 demonstrates a summary of results of retrotransposon integration and expression across cell types.

FIG. 39 shows various applications of the technology described herein, including but not limited to use of CART cells, NK cells, neurons and other cells for cell therapy, and use of in vivo applications in including but not limited to gene therapy, gene editing, transcription regulation, and genome engineering.

FIG. 40 depicts exemplary flow cytometry data showing sorting and enriching GFP+ 293T cells electroporated with 2000 ng/μL LINE1-GFP mRNA. The first panel shows flow cytometry data for mock electroporated cells in the absence of LINE1-GFP mRNA. The second panel shows flow cytometry data collected 5 days post electroporation for unsorted cells electroporated with LINE1-GFP mRNA. The GFP+ cells from the second panel were sorted and the flow cytometry data are shown in the third panel. The GFP+ cells from the third panel were cultured for 9 days post sorting and resorted using 10{circumflex over ( )}3 or 10{circumflex over ( )}4 GFP fluorescence intensity gate. The fourth panel shows flow cytometry data for cells resorted using GFP+ at 10{circumflex over ( )}3 GFP gate collected 4 days after resorting. The fifth panel shows flow cytometry data for cells resorted using GFP+ at 10{circumflex over ( )}3 GFP gate collected 4 days after resorting.

FIG. 41A shows a standard curve for GFP (NB2 plasmid) and a housekeeping gene (FAU) for evaluating genomic integration of GFP-encoding nucleic acid per cell using quantitative PCR.

FIG. 41B shows results of an exemplary graph depicting interpolation of the standard curves of FIG. 41A for quantitation of genomic integration.

FIG. 41C shows the number of the GFP gene integrated into genome of 293T cells following LINE1-GFP mRNA electroporation and double sorting as shown in FIG. 40. The average number of GFP integrations per cell when gated at 10{circumflex over ( )}3 GFP+ cells and at 10{circumflex over ( )}4 GFP+ cells according to qPCR are shown.

FIG. 42 depicts exemplary flow cytometry data showing GFP+ 293T cells electroporated with the indicated titrated amounts of LINE1-GFP mRNA, in ng/μL in electroporation solution, after culturing for 3 days post-electroporation.

FIG. 43 depicts exemplary flow cytometry data showing GFP+ 293T cells electroporated with the indicated titrated amounts of LINE1-GFP mRNA, in ng/μL in electroporation solution, after culturing for 5 days post-electroporation.

FIG. 44 depicts exemplary flow cytometry data showing GFP+ 293T cells electroporated with the indicated titrated amounts of LINE1-GFP mRNA, in ng/μL in electroporation solution, after culturing for 7 days post-electroporation.

FIG. 45 shows a graph of the number of GFP integrations per genome of 293T cells electroporated with the indicated titrated amounts of LINE1-GFP mRNA, in ng/μL in electroporation solution, according to qPCR after culturing for 3, 5 or 7 days post-electroporation according to FIGS. 42-44 (top) and a graph of the integration kinetics (bottom) according to the data from FIGS. 42-44.

FIG. 46 depicts exemplary flow cytometry data (right) showing GFP+ K562 cells electroporated with the indicated titrated amounts of LINE1-GFP mRNA, in ng/μL in electroporation solution, after culturing for 6 days post-electroporation, and a graph of the number of GFP integrations per genome according to qPCR (left).

FIG. 47 depicts exemplary flow cytometry data (top) showing GFP+ human primary monocytes electroporated with the indicated titrated amounts of LINE1-GFP mRNA after culturing for 3 days post-electroporation, and a graph of the number of GFP integrations per genome according to qPCR (bottom).

FIG. 48 depicts exemplary flow cytometry data (bottom) showing GFP+ 293T cells electroporated with 2000 ng/μL LINE1-GFP mRNA and 100 ng/μL, 200 ng/μL or 300 ng/μL of an siRNA targeting BRCA1 (siBRCA1) after culturing for 4 days post-electroporation and a graph of the number of GFP integrations per genome according to qPCR (top).

FIG. 49 depicts exemplary flow cytometry data (bottom) showing GFP+ 293T cells electroporated with 2000 ng/μL LINE1-GFP mRNA and 100 ng/μL of an siRNA targeting RNASEL (siRNASEL), ADAR1 (siADAR1), or ADAR2 (siADAR2) after culturing for 6 days post-electroporation and a graph of the number of GFP integrations per genome according to qPCR (top).

FIG. 50 depicts exemplary flow cytometry data (bottom) showing GFP+293T cells electroporated with 2000 ng/μL LINE1-GFP mRNA and 100 ng/μL of an siRNA targeting APOBEC3C (siAPOBEC3C) or FAM208A (siFAM208A) after culturing for 6 days post-electroporation and a graph of the number of GFP integrations per genome according to qPCR (top).

FIG. 51 depicts exemplary flow cytometry data (bottom) showing GFP+293T cells electroporated with 1000 ng/μL or 1500 ng/μL LINE1-GFP mRNA and an siRNA cocktail with 25 ng/μL, 50 ng/μL or 75 ng/μL of each siRNA targeting RNASEL (siRNASEL), ADAR1 (siADAR1), ADAR2 (siADAR2) and BRCA1 (siBRCA1) after culturing for 6 days post-electroporation and a graph of the number of GFP integrations per genome according to qPCR (top).

FIG. 52 depicts exemplary flow cytometry data (bottom) showing GFP+ K562 cells electroporated with 1000 ng/μL LINE1-GFP mRNA and an siRNA cocktail with 25 ng/μL, 50 ng/μL or 75 ng/4 of each siRNA targeting RNASEL (siRNASEL), ADAR1 (siADAR1), ADAR2 (siADAR2) and BRCA1 (siBRCA1) after culturing for 5 days post-electroporation and a graph of the number of GFP integrations per cell according to qPCR (top).

FIG. 53 depicts a schematic showing exemplary locations of extraneous nuclear localization sequences (NLS) and exemplary ORF1p and ORF2p mutations of an exemplary LINE1-GFP mRNA construct.

DETAILED DESCRIPTION

The present invention arises in part from the exciting discovery that a polynucleotide could be designed and developed to accomplish transfer and integration of a genetic cargo (e.g., large genetic cargo) into the genome of a cell. In some embodiments, the polynucleotide comprises (i) a genetic material for stable expression, and (ii) a self-integrating genomic integration machinery that allows stable integration of the genetic material into a cell by non-viral means, that is both safe and efficacious. Moreover, the genetic material may be integrated at a locus other than a ribosomal locus; the genetic material may be integrated site-specifically; and/or the integrated genetic material appear to express without triggering a cell's natural silencing machinery.

Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) revolutionized the molecular biology field and has developed into a potent gene editing too. It utilizes homology-directed repair (HDR) and can be directed to a genomic site. CRISPR/Cas9 is a naturally occurring RNA-guided endonuclease. While the CRISPR/Cas9 system has demonstrated great promise for site-specific gene editing and other applications, there are several factors that influence its efficacy which must be addressed, especially if it is to be used for in vivo human gene therapy. These factors include target DNA site selection, sgRNA design, off-target cutting, incidence/efficiency of HDR vs. NHEJ, Cas9 activity, and the method of delivery. Delivery remains the major obstacle for use of CRISPR for in vivo applications. Zinc finger nucleases ZFNs are a fusion protein of Cys2-His2 zinc finger proteins (ZFPs) and a non-specific DNA restriction enzyme derived from FokI endonucleases. Challenges with ZFPs include design and engineering of the ZFP for high-affinity binding of the desired sequence, which is non-trivial. Also, not all sequences are available for ZFP binding, so site selection is limited. Another significant challenge is off-target cutting. Transcription activator-like effector nucleases (TALENs) are a fusion protein comprised of a TALE and a FokI nuclease. While off-target cutting remains a concern, TALENs have been shown in one side-by-side comparison study to be more specific and less cytotoxic than ZFNs. However, TALENs are substantially larger, and the cDNA encoding TALEN only is 3 kb. This makes delivery of a pair of TALENs more challenging than a pair of ZFNs due to delivery vehicle cargo size limitations. Further, packaging and delivery of TALENs in some viral vectors may be problematic due to the high level of repetition in the TALENs sequence. A mutant Cas9 system, a fusion protein of inactive dCas9 and a FokI nuclease dimer increase specificity and reduce off-target cutting, the number of potential target sites is lower due to PAM and other sgRNA design constraints.

The present invention addresses the problems described above by providing new, effective and efficient compositions comprising transposon-based vectors for providing therapy, including gene therapy, to animals and humans. The present invention provides methods of using these compositions for providing therapy to animals and humans. These transposon-based vectors can be used in the preparation of a medicament useful for providing a desired effect to a recipient following administration. Gene therapy includes, but is not limited to, introduction of a gene, such as an exogenous gene, into an animal using a transposon-based vector. These genes may serve a variety of functions in the recipient such as coding for the production of nucleic acids, for example RNA, or coding for the production of proteins and peptides. The present invention can facilitate efficient incorporation of the polynucleotide sequences, including the genes of interest, promoters, insertion sequences, poly A and any regulatory sequences. The invention is based on the finding that human LINE-1 elements are capable of retrotransposition in human cells as well as cells of other animal species and can be manipulated in a versatile manner to achieve efficient delivery and integration of a genetic cargo into the genome of a cell. Such LINE-1 elements have a variety of uses in human and animal genetics including, but not limited to, uses in diagnosis and treatment of genetic disorders and in cancer. The LINE-1 elements of the invention are also useful for the treatment of various phenotypic effects of various diseases. For example, LINE-1 elements may be used for transfer of DNA encoding anti-tumorigenic gene products into cancer cells. Other uses of the LINE-1 elements of the invention will become apparent to the skilled artisan upon a reading of the present specification.

In general, a human LINE-1 element comprises a 5′ UTR with an internal promoter, two non-overlapping reading frames (ORF1 and ORF2), a 200 bp 3′ UTR and a 3′ poly A tail. The LINE-1 retrotransposon can also comprise an endonuclease domain at the LINE-1 ORF2 N-terminus. The finding that LINE-1 encodes an endonuclease demonstrates that the element is capable of autonomous retrotransposition. LINE-1 is a modular protein that contains non-overlapping functional domains which mediate its reverse transcription and integration. In some embodiments, the sequence specificity of the LINE-1 endonuclease itself can be altered or the LINE-1 endonuclease can be replaced with another site-specific endonuclease.

The LINE-1 retrotransposon may be manipulated using recombinant DNA technology to comprise and/or be contiguous with, other DNA elements which render the retrotransposon suitable for insertion of substantial lengths (up to 1 kb, or greater than 1 kb) of heterologous or homologous DNA into the genome of a cell. The LINE-1 retrotransposon may also be manipulated using the same type of technology such that insertion of the DNA into the genome of a cell is site-directed (site into which such DNA is inserted is known). Alternatively, the LINE-1 retrotransposon may be manipulated such that the insertion site of the DNA is random. The retrotransposon may also be manipulated to effect insertion of a desired DNA sequence into regions of DNA which are normally transcriptionally silent, wherein the DNA sequence is expressed in a manner such that it does not disrupt the normal expression of genes in the cell. In some embodiments, the integration or retrotransposition is in the trans orientation. In some embodiments, the integration or retrotransposition occurs in the cis orientation.

Since LINE-1 is native to human cells, when the constructs are placed into human cells, they should not be rejected by the immune system as foreign. In addition, the mechanism of LINE-1 retro-integration ensures that only one copy of the gene is integrated at any specific chromosomal location. Accordingly, there is a copy number control built into the system. In contrast, gene transfer procedures using ordinary plasmids offer little or no control regarding copy number and often result in complex arrays of DNA molecules tandemly integrated into the same genomic location.

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

In this application, the use of “or” means “and/or” unless stated otherwise. The terms “and/or” and “any combination thereof” and their grammatical equivalents as used herein, may be used interchangeably. These terms may convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof” may mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” may be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

The term “about” or “approximately” may mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification may be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure may be used to achieve methods of the present disclosure.

Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the disclosure can also be implemented in a single embodiment.

Applications of the present disclosure encompasses, but are not limited to methods and compositions related to expression of an exogenous nucleic acid in a cell. In some embodiments, the exogenous nucleic acid is configured for stable integration in the genome of a cell, such as a myeloid cell. In some embodiments, the stable integration of the exogenous nucleic acid may be at specific targets within the genome. In some embodiments, the exogenous nucleic acid comprises one or more coding sequences. In some embodiments, the exogenous nucleic acid may comprise one or more coding comprising a nucleic acid sequence encoding an immune receptor. In some embodiments, the present disclosure provides methods and compositions for a stable incorporation of a nucleic acid encoding a transmembrane receptor implicated in an immune response function (e.g. a phagocytic receptor or synthetic chimeric antigen receptor) into human macrophage or dendritic cell or a suitable myeloid cell or a myeloid precursor cell. An exogenous nucleic acid can refer to a nucleic acid that was not originally in a cell and is added from outside the cell, irrespective of whether it comprises a sequence that may already be present in the cell endogenously. An exogenous nucleic acid may be a DNA or an RNA molecule. An exogenous nucleic acid may comprise a sequence encoding a transgene. An exogenous nucleic acid may encode a recombinant protein, such as a recombinant receptor, or a chimeric antigen receptor (CAR). An exogenous nucleic acid may be referred to as a “genetic cargo” in the context of the exogenous nucleic acid being delivered inside a cell. The genetic cargo may be a DNA or an RNA. Genetic material can generally be delivered inside a cell ex vivo by a few different known techniques using either chemical (CaCl₂)-medicated transfection), or physical (electroporation), or biological (e.g. viral infection or transduction) means.

In one aspect, provided herein are methods and compositions for delivery inside a cell, for example a myeloid cell and stable incorporation of one or more nucleic acids, comprising nucleic acid sequences encoding one or more proteins, wherein the stable incorporation may be via non-viral mechanisms. In some embodiments, the delivery of a nucleic acid composition into a myeloid cell is via a non-viral mechanism. In some embodiments, the delivery of the nucleic acids may further bypass plasmid mediated delivery. A “plasmid,” as used herein, refers to a non-viral expression vector, e.g., a nucleic acid molecule that encodes for genes and/or regulatory elements necessary for the expression of genes. A “viral vector,” as used herein, refers to a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors.

In some embodiments, provided herein is a method of delivering a composition inside a cell, such as in a myeloid cell, the composition comprising one or more nucleic acid sequences encoding one or more proteins, wherein the one or more nucleic acid sequences is an RNA. In some embodiments, the RNA is mRNA. In some embodiments, one or more mRNA comprising one or more nucleic acid sequences are delivered. In some embodiments, the one or more mRNA may comprise at least one modified nucleotide. The term “nucleotide,” as used herein, refers to a base-sugar-phosphate combination. A nucleotide may comprise a synthetic nucleotide. A nucleotide may comprise a synthetic nucleotide analog. Nucleotides may be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, or derivatives thereof. Such derivatives may include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling may also be carried out with quantum dots. Detectable labels may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,NcN′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides may include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAN 1RA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, TR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-1 4-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides may also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-cICTP, biotin-14-dCTP), and biotin-dUTP (e.g. biotin-11-dUTP, biotin-1.6-dUTP, biotin-20-dUTP).

The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may exist in a cell-free environment. A polynucleotide may be a gene or fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure, and may perform any function, known or unknown. A polynucleotide may comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of modified nucleotides or analogs include: pseudouridine, 5-bromouracil, 5-methylcytosine, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

In some embodiments, the nucleic acid composition may comprise one or more mRNA, comprising at least one mRNA encoding a transmembrane receptor implicated in an immune response function (e.g. a phagocytic receptor or synthetic chimeric antigen receptor) into human macrophage or dendritic cell or a suitable myeloid cell or a myeloid precursor cell. In some embodiments, the nucleic acid composition comprises one or more mRNA, and one or more lipids for delivery of the nucleic acid into a cell of hematopoietic origin, such as a myeloid cell or a myeloid cell precursor cell. In some embodiments, the one or more lipids may form a liposomal complex.

As used herein, the composition described herein may be used for delivery inside a cell. A cell may originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g. cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g. kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell may not be originating from a natural organism (e.g. a cell may be a synthetically made, sometimes termed an artificial cell). In some embodiments, the cell referred to herein is a mammalian cell. In some embodiments, the cell is a human cell. The methods and compositions described herein relates to incorporating a genetic material in a cell, more specifically a human cell, wherein the human cell can be any human cell. As used herein, a human cell may be of any origin, for example, a somatic cell, a neuron, a fibroblast, a muscle cell, an epithelial cell, a cardiac cell, or a hematopoietic cell. The methods and compositions described herein can also be applicable to and useful for incorporating exogenous nucleic acid in hard-to-transfect human cell. The methods are simple and universally applicable, once a suitable exogenous nucleic acid construct has been designed and developed. The methods and compositions described herein are applicable to incorporate an exogenous nucleic acid in a cell ex vivo. In some embodiments, the compositions may be applicable for systemic administration in an organism, where the nucleic acid material in the composition may be taken up by a cell in vivo, whereupon it is incorporated in cell in vivo.

In some embodiments, the methods and compositions described herein may be directed to incorporating an exogenous nucleic acid in a human hematopoietic cell, for example, a human cell of hematopoietic origin, such as a human myeloid cell or a myeloid cell precursor. However, the methods and compositions described herein can be used or made suitable for use in any biological cell with minimum modifications. Therefore, a cell as may refer to any cell that is a basic structural, functional and/or biological unit of a living organism.

In one aspect, provided herein are methods and compositions for utilizing transposable elements for stable incorporation of one or more nucleic acids into the genome of a cell, where the cell is a member of a hematopoietic cells, for example a myeloid cell. In some embodiments, the one or more nucleic acids comprise at least one nucleic acid sequence encoding a transmembrane receptor protein having a role in immune response. In some embodiments, the methods and compositions are directed to using a retrotransposable element for incorporating one or more nucleic acid sequences into a myeloid cell. The nucleic acid composition may comprise one or more nucleic sequences, such as a gene, where the gene is a transgene. The term “gene,” as used herein, refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and may include 5′ and 3′ ends. In some uses, the term encompasses the transcribed sequences, including 5′ and 3′ untranslated regions (5′-UTR and 3′-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene may refer to an “exogenous gene” or a non-native gene. A non-native gene may refer to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene not in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence).

The term “transgene” refers to any nucleic acid molecule that is introduced into a cell, that may be intermittently termed herein as a recipient cell. The resultant cell after receiving a transgene may be referred to a transgenic cell. A transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism or cell, or may represent a gene homologous to an endogenous gene of the organism or cell. In some cases, transgenes include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks an expression control element, such as a promoter that drives transcription). Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. “Up-regulated,” with reference to expression, refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene may occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene may occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. Where a transfected gene is required to be expressed, the application envisages the use of codon-optimized sequences. An example of a codon optimized sequence may be a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal. Codon optimization for a host species other than human, or for codon optimization for specific organs is known. In some embodiments, the coding sequence encoding a protein may be codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally reflect the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.

A “multicistronic transcript” as used herein refers to an mRNA molecule that contains more than one protein coding region, or cistron. A mRNA comprising two coding regions is denoted a “bicistronic transcript.” The “5′-proximal” coding region or cistron is the coding region whose translation initiation codon (usually AUG) is closest to the 5′ end of a multicistronic mRNA molecule. A “5′-distal” coding region or cistron is one whose translation initiation codon (usually AUG) is not the closest initiation codon to the 5′ end of the mRNA.

The terms “transfection” or “transfected” refer to introduction of a nucleic acid into a cell by non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. See, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.

The term “promoter,” as used herein, refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the disclosure include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter may be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. A “constitutive promoter” is one that is capable of initiating transcription in nearly all tissue types, whereas a “tissue-specific promoter” initiates transcription only in one or a few particular tissue types. An “inducible promoter” is one that initiates transcription only under particular environmental conditions, developmental conditions, or drug or chemical conditions. Exemplary inducible promoter may be a doxycycline or a tetracycline inducible promoter. Tetracycline regulated promoters may be both tetracycline inducible or tetracycline repressible, called the tet-on and tet-off systems. The tet regulated systems rely on two components, i.e., a tetracycline-controlled regulator (also referred to as transactivator) (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls expression of a downstream cDNA, in a tetracycline-dependent manner. tTA is a fusion protein containing the repressor of the Tn10 tetracycline-resistance operon of Escherichia coli and a carboxyl-terminal portion of protein 16 of herpes simplex virus (VP16). The tTA-dependent promoter consists of a minimal RNA polymerase II promoter fused to tet operator (tetO) sequences (an array of seven cognate operator sequences). This fusion converts the tet repressor into a strong transcriptional activator in eukaryotic cells. In the absence of tetracycline or its derivatives (such as doxycycline), tTA binds to the tetO sequences, allowing transcriptional activation of the tTA-dependent promoter. However, in the presence of doxycycline, tTA cannot interact with its target and transcription does not occur. The tet system that uses tTA is termed tet-OFF, because tetracycline or doxycycline allows transcriptional down-regulation. In contrast, in the tet-ON system, a mutant form of tTA, termed rtTA, has been isolated using random mutagenesis. In contrast to tTA, rtTA is not functional in the absence of doxycycline but requires the presence of the ligand for transactivation. The term “exon” refers to a nucleic acid sequence found in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to contribute contiguous sequence to a mature mRNA transcript. The term “intron” refers to a sequence present in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to not encode part of or all of an expressed protein, and which, in endogenous conditions, is transcribed into RNA (e.g. pre-mRNA) molecules, but which is spliced out of the endogenous RNA (e.g. the pre-mRNA) before the RNA is translated into a protein.

The term “splice acceptor site” refers to a sequence present in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to be the acceptor site during splicing of pre-mRNA, which may include identified and unidentified natural and artificially derived or derivable splice acceptor sites.

An “internal ribosome entry site” or “IRES” refers to a nucleotide sequence that allows for 5′-end/cap-independent initiation of translation and thereby raises the possibility to express 2 proteins from a single messenger RNA (mRNA) molecule. IRESs are commonly located in the 5′ UTR of positive-stranded RNA viruses with uncapped genomes. Another means to express 2 proteins from a single mRNA molecule is by insertion of a 2A peptide(-like) sequence in between their coding sequence. 2A peptide(-like) sequences mediate self-processing of primary translation products by a process variously referred to as “ribosome skipping”, “stop-go” translation and “stop carry-on” translation. 2A peptide(-like) sequences are present in various groups of positive- and double-stranded RNA viruses including Picornaviridae, Flaviviridae, Tetraviridae, Dicistroviridae, Reoviridae and Totiviridae.

The term “2A peptide” refers to a class of 18-22 amino-acid (AA)-long viral oligopeptides that mediate “cleavage” of polypeptides during translation in eukaryotic cells. The designation “2A” refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. The first discovered 2A was F2A (foot-and-mouth disease virus), after which E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A) were also identified. The mechanism of 2A-mediated “self-cleavage” is believed to be ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A sequence. 2A peptide(-like) sequences mediate self-processing of primary translation products by a process variously referred to as “ribosome skipping”, “stop-go” translation and “stop carry-on” translation. 2A peptide(-like) sequences are present in various groups of positive- and double-stranded RNA viruses including Picornaviridae, Flaviviridae, Tetraviridae, Dicistroviridae, Reoviridae and Totiviridae.

As used herein, the term “operably linked” refers to a functional relationship between two or more segments, such as nucleic acid segments or polypeptide segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.

The term “termination sequence” refers to a nucleic acid sequence which is recognized by the polymerase of a host cell and results in the termination of transcription. The termination sequence is a sequence of DNA that, at the 3′ end of a natural or synthetic gene, provides for termination of mRNA transcription or both mRNA transcription and ribosomal translation of an upstream open reading frame. Prokaryotic termination sequences commonly comprise a GC-rich region that has a two-fold symmetry followed by an AT-rich sequence. A commonly used termination sequence is the T7 termination sequence. A variety of termination sequences are known in the art and may be employed in the nucleic acid constructs of the present invention, including the TINT3, TL13, TL2, TR1, TR2, and T6S termination signals derived from the bacteriophage lambda, and termination signals derived from bacterial genes, such as the trp gene of E. coli.

The terms “polyadenylation sequence” (also referred to as a “poly A site” or “poly A sequence”) refers to a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly A tail are typically unstable and rapidly degraded. The poly A signal utilized in an expression vector may be “heterologous” or “endogenous”. An endogenous poly A signal is one that is found naturally at the 3′ end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3′ of another gene, e.g., coding sequence for a protein. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237 bp BamHI/BclI restriction fragment and directs both termination and polyadenylation; numerous vectors contain the SV40 poly A signal. Another commonly used heterologous poly A signal is derived from the bovine growth hormone (BGH) gene; the BGH poly A signal is also available on a number of commercially available vectors. The poly A signal from the Herpes simplex virus thymidine kinase (HSV tk) gene is also used as a poly A signal on a number of commercial expression vectors. The polyadenylation signal facilitates the transportation of the RNA from within the cell nucleus into the cytosol as well as increases cellular half-life of such an RNA. The polyadenylation signal is present at the 3′-end of an mRNA.

The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, refer to a sequence that is complementary to and hybridizable to the given sequence. In some cases, a sequence hybridized with a given nucleic acid is referred to as the “complement” or “reverse-complement” of the given molecule if its sequence of bases over a given region is capable of complementarily binding those of its binding partner, such that, for example, A-T, A-U, G-C, and G-U base pairs are formed. In general, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred (e.g. thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art) to hybridization with non-target sequences during a hybridization reaction. Typically, hybridizable sequences share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity. Sequence identity, such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/embossneedle/nucleotide.html), the BLAST algorithm (see e.g. the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see e.g. the EMBOSS Water aligner available at www.ebi.ac.ukaools/psa/emboss_water/nucleotide html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of a chosen algorithm, including default parameters.

Complementarity may be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids may mean that the two nucleic acids may form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. Substantial or sufficient complementary may mean that, a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions may be predicted by using the sequences and standard mathematical calculations to predict the melting temperature (Tm) of hybridized strands, or by empirical determination of Tm by using routine methods.

“Transposons” as used herein are segments within the chromosome that can translocate within the genome, also known as “jumping gene”. There are two different classes of transposons: class 1, or retrotransposons, that mobilize via an RNA intermediate and a “copy-and-paste” mechanism, and class II, or DNA transposons, that mobilize via excision integration, or a “cut-and-paste” mechanism (Ivics Nat Methods 2009). Bacterial, lower eukaryotic (e.g. yeast) and invertebrate transposons appear to be largely species specific, and cannot be used for efficient transposition of DNA in vertebrate cells. “Sleeping Beauty” (Ivics Cell 1997), was the first active transposon that was artificially reconstructed by sequence shuffling of inactive TEs from fish. This made it possible to successfully achieve DNA integration by transposition into vertebrate cells, including human cells. Sleeping Beauty is a class II DNA transposon belonging to the Tcl/mariner family of transposons (Ni Genomics Proteomics 2008). In the meantime, additional functional transposons have been identified or reconstructed from different species, including Drosophila, frog and even human genomes, that all have been shown to allow DNA transposition into vertebrate and also human host cell genomes. Each of these transposons have advantages and disadvantages that are related to transposition efficiency, stability of expression, genetic payload capacity etc. Exemplary class II transposases that have been created include Sleeping Beauty, PiggyBac, Frog Prince, Himarl, Passport, Minos, hAT, Toll, To12, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmarl.

“Heterologous” as used herein, includes molecules such as DNA and RNA which may not naturally be found in the cell into which it is inserted. For example, when mouse or bacterial DNA is inserted into the genome of a human cell, such DNA is referred to herein as heterologous DNA. In contrast, the term “homologous” as used herein, denotes molecules such as DNA and RNA that are found naturally in the cell into which it is inserted. For example, the insertion of mouse DNA into the genome of a mouse cell constitutes insertion of homologous DNA into that cell. In the latter case, it is not necessary that the homologous DNA be inserted into a site in the cell genome in which it is naturally found; rather, homologous DNA may be inserted at sites other than where it is naturally found, thereby creating a genetic alteration (a mutation) in the inserted site.

A “transposase” is an enzyme that is capable of forming a functional complex with a transposon end-containing composition (e.g., transposons, transposon ends), and catalyze insertion or transposition of the transposon end-containing composition into double stranded DNA which is incubated with an in vitro transposon reaction. The term “transposon end” means a double-stranded DNA that contains the nucleotide sequences (the “transposon end sequences”) necessary to form the complex with the transposase or integrase enzyme that is functional in an in vitro transposition reaction.

A transposon end forms a complex or a synaptic complex or a transposon complex or a transposon composition with a transposase or integrase that recognizes and binds to the transposon end, and which complex is capable of inserting or transposing the transposon end into target DNA with which it is incubated in an in vitro transposition reaction. A transposon end exhibits two complementary sequences consisting of a transferred transposon end sequence or transferred strand and a non-transferred transposon end sequence, or non-transferred strand For example, one transposon end that forms a complex with a hyperactive Tn5 transposase that is active in an in vitro transposition reaction comprises a transferred strand that exhibits a transferred transposon end sequence as follows: 5′ AGATGTGTATAAGAGACAG 3′ (SEQ ID NO: 51), and a non-transferred strand that exhibits a “non-transferred transposon end sequence” as follows: 5′ CTGTCTCTTATACACATCT 3 (SEQ ID NO: 52)′. The 3′-end of a transferred strand is joined or transferred to target DNA in an in vitro transposition reaction. The non-transferred strand, which exhibits a transposon end sequence that is complementary to the transferred transposon end sequence, is not joined or transferred to the target DNA in an in vitro transposition reaction.

In some embodiments, the transferred strand and non-transferred strand are covalently joined. For example, in some embodiments, the transferred and non-transferred strand sequences are provided on a single oligonucleotide, e.g., in a hairpin configuration. As such, although the free end of the non-transferred strand is not joined to the target DNA directly by the transposition reaction, the non-transferred strand becomes attached to the DNA fragment indirectly, because the non-transferred strand is linked to the transferred strand by the loop of the hairpin structure. As used herein an “cleavage domain” refers to a nucleic acid sequence that is susceptible to cleavage by an agent, e.g., an enzyme.

A “restriction site domain” means a tag domain that exhibits a sequence for the purpose of facilitating cleavage using a restriction endonuclease. For example, in some embodiments, the restriction site domain is used to generate di-tagged linear ssDNA fragments. In some embodiments, the restriction site domain is used to generate a compatible double-stranded 5′-end in the tag domain so that this end can be ligated to another DNA molecule using a template-dependent DNA ligase. In some embodiments, the restriction site domain in the tag exhibits the sequence of a restriction site that is present only rarely, if at all, in the target DNA (e.g., a restriction site for a rare-cutting restriction endonuclease such as NotI or AscI).

As used herein, the term “recombinant nucleic acid molecule” refers to a recombinant DNA molecule or a recombinant RNA molecule. A recombinant nucleic acid molecule is any nucleic acid molecule containing joined nucleic acid molecules from different original sources and not naturally attached together. Recombinant RNA molecules include RNA molecules transcribed from recombinant DNA molecules. A recombinant nucleic acid may be synthesized in the laboratory. A recombinant nucleic acid can be prepared by using recombinant DNA technology by using enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. A recombinant DNA may be transcribed in vitro, to generate a messenger RNA (mRNA), the recombinant mRNA may be isolated, purified and used to transfect a cell. A recombinant nucleic acid may encode a protein or a polypeptide. A recombinant nucleic acid, under suitable conditions, can be incorporated into a living cell, and can be expressed inside the living cell. As used herein, “expression” of a nucleic acid usually refers to transcription and/or translation of the nucleic acid. The product of a nucleic acid expression is usually a protein but can also be an mRNA. Detection of an mRNA encoded by a recombinant nucleic acid in a cell that has incorporated the recombinant nucleic acid, is considered positive proof that the nucleic acid is “expressed” in the cell. The process of inserting or incorporating a nucleic acid into a cell can be via transformation, transfection or transduction. Transformation is the process of uptake of foreign nucleic acid by a bacterial cell. This process is adapted for propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in a laboratory setting. Transfection is the forced introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Just to make life confusing, ‘transfection’ also refers to the introduction of bacteriophage into bacterial cells. ‘Transduction’ is mostly used to describe the introduction of recombinant viral vector particles into target cells, while ‘infection’ refers to natural infections of humans or animals with wild-type viruses.

A “stem-loop” sequence refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to hybridize and form a stem and the regions of non-complementarity that bulges into a loop. The stem may comprise mismatches or bulges.

The term “vector” refers to a nucleic acid molecule capable of transporting or mediating expression of a heterologous nucleic acid. A “vector sequence” as used herein, refers to a sequence of nucleic acid comprising at least one origin of replication and at least one selectable marker gene. Vectors capable of directing the expression of genes and/or nucleic acid sequence to which they are operatively linked are referred to herein as “expression vectors”.

A plasmid is a species of the genus encompassed by the term “vector.” In general, expression vectors of utility are often in the form of “plasmids” which refer to circular double stranded DNA molecules which, in their vector form are not bound to the chromosome, and typically comprise entities for stable or transient expression of the encoded DNA. Other expression vectors that can be used in the methods as disclosed herein include, but are not limited to plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages or viral vectors, and such vectors can integrate into the host's genome or replicate autonomously in the cell. A vector can be a DNA or RNA vector. Other forms of expression vectors known by those skilled in the art which serve the equivalent functions can also be used, for example, self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome. Exemplary vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. A safe harbor locus is a region within the genome where additional exogenous or heterologous nucleic acid sequence can be inserted, and the host genome is able to accommodate the inserted genetic material. Exemplary safe harbor sites include but are not limited to: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site, and TIGRE site. For example, the heterologous nucleic acid described in this disclosure may be integrated at one or more sites in the genome of the cell, wherein the one or more locations is selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site, and TIGRE site. In some embodiments, the nucleic acid cargo comprising the transgene may be delivered to a R2D locus.

In some embodiments, the nucleic acid cargo comprising the transgene may be delivered to the genome in an intergenic or intragenic region. In some embodiments the nucleic acid cargo comprising the transgene is integrated into the genome 5′ or 3′ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous active gene. In some embodiments the nucleic acid cargo comprising the transgene is integrated into the genome 5′ or 3′ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous promoter or enhancer. In some embodiments the nucleic acid cargo comprising the transgene is 50-50,000 base pairs, e.g., between 50-40,000 bp, between 500-30,000 bp between 500-20,000 bp, between 100-15,000 bp, between 500-10,000 bp, between 50-10,000 bp, between 50-5,000 bp. In some embodiments the nucleic acid cargo comprising the transgene is less than 1,000, 1,300, 1500, 2,000, 3,000, 4,000, 5,000, or 7,500 nucleotides in length.

L1 and Non-L1 Retrotransposon Systems

Retrotransposons can contain transposable elements that are active participants in reorganizing their resident genomes. Broadly, retrotransposons can refer to DNA sequences that are transcribed into RNA and translated into protein and have the ability to reverse-transcribe themselves back into DNA. Approximately 45% of the human genome is comprised of sequences that result from transposition events. Retrotransposition occasionally generates target site deletions or adds non-retrotransposon DNA to the genome by processes termed 5′- and 3′-transduction. Recombination between non-homologous retrotransposons causes deletions, duplications or rearrangements of gene sequence. Ongoing retrotransposition can generate novel splice sites, polyadenylation signals and promoters, and so builds new transcription modules.

Generally, retrotransposons may be grouped into two classes, the retrovirus-like LTR retrotransposons, and the non-LTR elements such as human L1 elements, Neurospora TAD elements (Kinsey, 1990, Genetics 126:317-326), I factors from Drosophila (Bucheton et al., 1984, Cell 38:153-163), and R2Bm from Bombyx mori (Luan et al., 1993, Cell 72: 595-605). These two types of retrotransposons are structurally different and also retrotranspose using radically different mechanisms. Exemplary, non-limiting examples of LINE-encoded polypeptides are found in GenBank Accession Nos. AAC51261, AAC51262, AAC51263, AAC51264, AAC51265, AAC51266, AAC51267, AAC51268, AAC51269, AAC51270, AAC51271, AAC51272, AAC51273, AAC51274, AAC51275, AAC51276, AAC51277, AAC51278 and AAC51279.

The decision to focus on LINE-1 to develop into a system as described in the disclosure for a number of reasons at least some of which are exemplified below: (a) LINE-1 (or L1-) elements are autonomous as they encode all of the machinery alone to complete this reverse transcription and integration process; (b) L1 elements are abundant in the human genome, such that these elements may be considered as a naturalized element of the genome; (c) L1 retrotransposon retrotransposes its own mRNA with high degree of specificity, compared to other mRNAs floating around in the cells.

The L1 expresses a 6-kb bicistronic RNA that encodes the 40 kDa Open Reading Frame-1 RNA-binding protein (ORF1p) of essential but uncertain function, and a 150 kDa ORF2 protein with endonuclease and reverse transcriptase (RT) activities. L1 retrotransposition is a complex process involving transcription of the L1, transport of its RNA to the cytoplasm, translation of the bicistronic RNA, formation of a ribonucleoprotein (RNP) particle, its re-import to the nucleus and target-primed reverse transcription at the integration site. A few transcription factors that interact with L1s have been identified. Transcribed L1 RNA forms an RNP in cis with the proteins that are translated from the transcript. L1 integrates into genomic DNA by target-site primer reverse transcription (TPRT) by ORF2p cleavage at the 5′-TTTT-3′ where a poly A sequence of L1 RNA anneals and primes reverse transcriptase (RT) activity to make L1 cDNA.

Other mobile elements of the genome can “hijack” the L1 ORF for retrotransposition. For example, Alu elements are such mobile DNA elements that belong to the class of short interspersed elements (SINEs) that are non-autonomous retrotransposons and acquire trans-factors to integrate. Alu elements and SINE-1 elements can associate with the L1 ribonucleoproteins in trans to be also retrotransposed by ORF1p and ORF2p. Somewhat similar to the L1 RNA, the Alu element ends with a long A-run, often referred to as the A-tail, and it also has a smaller A-rich region (indicated by AA) separating the two halves of a diverged dimer structure. Alu elements are likely to have the internal components of an RNA polymerase III promoter (such as, commonly designated as an A box and a B box promoters), but they do not encode a terminator for RNA polymerase III. They may utilize a stretch of T nucleotides at various distances downstream of the Alu element to terminate a transcription. A typical Alu transcript encompasses the entire Alu, including the A-tail, and has a 3′ region that is unique for each locus. The Alu RNA folds into separate structures for each monomer unit. The RNA has been shown to bind the 7SL RNA SRP9 and 14 heterodimer, as well as poly A-binding protein (PABP). The poly A tail of Alu primes with T rich (TTTT) region of the genome and attracts ORF2p to bind to the primed region and cleaves at the T rich region via its endonuclease activity. The T-rich region primes reverse transcription by ORF2p on the 3′ A-tail region of the Alu element. This creates a cDNA copy of the body of the Alu element. A nick occurs by an unknown mechanism on the second strand and second-strand synthesis is primed. The new Alu element is then flanked by short direct repeats that are duplicates of the DNA sequence between the first and second nicks. Alu elements are extremely prevalent within RNA molecules, owing to their preference for gene-rich regions. A full-length Alu (˜300 bp) is derived from the signal recognition particle RNA 7SL and consists of two similar monomers with an A-rich linker in-between, A- and B-boxes present in the 5′ monomer, and a poly-A tail lacking the preceding polyadenylation signal resulting in an elongated tail (up to 100 bp in length). Alus can be transcribed by RNA polymerase III using the internal promoters within the A- and B-boxes; however, Alus contain no ORFs and therefore do not encode for protein products.

Other non-L1 transposons include SVAs and HERV-Ks. A full-length SVA (SINE-VNTR-Alu) element (˜2-3 kb) is a composite unit that contains a CCCTCT repeat, two Alu-like sequences, a VNTR, a SINE-R region with env (envelope) gene, the 3′ LTR of HERV-K10, and a polyadenylation signal followed by a poly-A tail. It is most likely that SVAs are transcribed by RNA polymerase II, although it is unknown whether SVA elements carry an internal promoter.

A full-length HERV-K element (˜9-10 kb) is comprised of ancient remnants of endogenous retroviral sequences and includes two flanking LTR regions surrounding three retroviral ORFs: (1) gag encoding the structural proteins of a retroviral capsid; (2) pol-pro encoding the enzymes: protease, RT, and integrase; and (3) env encoding proteins allowing for horizontal transfer. The LTR of HERV-K contains an internal, bidirectional promoter that appears to be under the transcriptional control of RNA polymerase II.

L1 retrotransposition and RNA binding can take place at or near poly-A tail. The 3′-UTR plays a role in the recognition of stringent-type LINE RNA of ORF1 protein (ORF1p). Stringent-type LINEs can contain a stem-loop structure located at the end of the 3′UTR. Branched molecules consisting of junctions between transposon 3′-end cDNA and the target DNA, as well as specific positioning of L1 RNA within ORF2 protein (ORF2p), were detected during initial stages of L1 retrotransposition in vitro. Secondary or tertiary RNA structure shared by L1 and Alu are likely to be responsible for recognition by and binding of ORF2, possibly along with a poly-A tail. In some embodiments, the stem-loop structure located downstream of the poly-A sequence correlates with cleavage intensity.

Mechanisms for restricting or resolving L1 integration have also evolved for the sake of maintaining genetic integrity and stability of the genome. Non-homologous end-joining repair proteins, such as XRCC1, Ku70 and DNA-PK, have been implicated in resolution of the L1 integrate at the time of insertion. In addition, the cell has evolved a number of proteins that stand against unrestricted retrotransposition, including the APOBEC3 family of cytosine deaminases, adenosine deaminase ADAR1, chromatin-remodeling factors and members of the piRNA pathway for post-transcription gene silencing that functions in the male germ line.

I. Compositions Comprising Nucleic Acid Constructs and Methods Involved for Stable Expression of Encoded Protein

Provided herein is a recombinant nucleic acid encoding one or more proteins for expression in a cell, such as a myeloid cell. In one embodiment, the recombinant nucleic acid is designed for stable expression of the one or more proteins or polypeptides encoded by the recombinant nucleic acid. In some embodiments, the stable expression is achieved by incorporation of recombinant nucleic acid within the genome of the cell.

It can be easily understood by one of skill in the art that the compositions and methods described herein can be utilized to design products in which the recombinant nucleic acid may comprise one or more sequences that do not translate as a protein or a polypeptide component, but may encode an oligonucleotide that can be a regulatory nucleic acid, such as an inhibitor oligonucleotide product, such as an activator oligonucleotide.

In one aspect, provided herein is a composition comprising a synthetic nucleic acid, comprising a nucleic acid sequence encoding a gene of interest and one or more retrotransposable elements to stably incorporate a non-endogenous nucleic acid into a cell. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a myeloid cell. In some embodiments, the cell is a precursor cell. In some embodiments, the cell is undifferentiated. In some embodiments, the cell has further differentiation potential. In some embodiments, the cell is not a stem cell.

A. LINE/Alu Retrotransposon Construct

In some embodiments, the present disclosure may utilize a retrotransposable system to stably incorporate into the genome and express a non-endogenous nucleic acid, where the non-endogenous nucleic acid comprises retrotransposable elements within the nucleic acid sequence. In some embodiments, the present disclosure may utilize a cell's endogenous retrotransposable system (e.g., proteins and enzymes), to stably express a non-endogenous nucleic acid in the cell. In some embodiments, the present disclosure may utilize a cell's endogenous retrotransposable system (e.g., proteins and enzymes, such as a LINE1 retrotransposition system), but may further express one or more components of the retrotransposable system to stably express a non-endogenous nucleic acid in the cell.

In some embodiments, a synthetic nucleic acid is provided herein, the synthetic nucleic acid encoding a transgene, and encoding one or more components for retrotransposition. The synthetic nucleic acid described herein is interchangeably termed as a nucleic acid construct, transgene or the exogenous nucleic acid.

In one aspect, provided herein is a method of integrating a nucleic acid sequence into a genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA into the cell, wherein the mRNA comprises: an insert sequence, wherein the insert sequence comprises an exogenous sequence, or a sequence that is a reverse complement of the exogenous sequence; a 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for a human ORF protein, and wherein the insert sequence is integrated into the genome of the cell.

In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for human ORF2p.

In one aspect, provided herein is a method for integrating a nucleic acid sequence into the genome of an immune cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence, wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and wherein the transgene sequence is integrated into the genome of the immune cell.

In one aspect, provided herein is a method for integrating a nucleic acid sequence into the genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; a 5′ UTR sequence, a sequence of a human retrotransposon downstream of the 5′ UTR sequence, and a 3′ UTR sequence downstream of the sequence of a human retrotransposon; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and/or a reverse transcriptase binding site, and wherein the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs, and wherein the insert sequence is integrated into the genome of the cell.

In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises an ORF2p binding site. In some embodiments, the ORF2p binding site is a poly A sequence in the 3′ UTR sequence.

In some embodiments, the mRNA comprises a sequence of a human retrotransposon. In some embodiments, the sequence of a human retrotransposon is downstream of the 5′ UTR sequence. In some embodiments, the sequence of a human retrotransposon is upstream of the 3′ UTR sequence.

In some embodiments, the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs. In some embodiments, the two ORFs are non-overlapping ORFs. In some embodiments, the two ORFs are ORF1 and ORF2. In some embodiments, the ORF1 encodes ORF1p and ORF2 encodes ORF2p.

In some embodiments, the sequence of a human retrotransposon comprises a sequence of a non-LTR retrotransposon. In some embodiments, the sequence of a human retrotransposon encodes comprises a LINE-1 retrotransposon. In some embodiments, the LINE-1 retrotransposon is a human LINE-1 retrotransposon. In some embodiments, the sequence of a human retrotransposon comprises a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the endonuclease and/or a reverse transcriptase is ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase domain. In some embodiments, the endonuclease and/or a reverse transcriptase is a minke whale endonuclease and/or a reverse transcriptase. In some embodiments, the sequence of a human retrotransposon comprises a sequence encoding ORF2p. In some embodiments, the insert sequence is integrated into the genome at a poly T site using specificity of an endonuclease domain of the ORF2p. In some embodiments, the poly T site comprises the sequence TTTTTA.

In some embodiments, (i) the sequence of a human retrotransposon comprises a sequence encoding ORF1p, (ii) the mRNA does not comprise a sequence encoding ORF1p, or (iii) the mRNA comprises a replacement of the sequence encoding ORF1p with a 5′ UTR sequence from the complement gene. In some embodiments, the mRNA comprises a first mRNA molecule encoding ORF1p, and a second mRNA molecule encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the mRNA is an mRNA molecule comprising a first sequence encoding ORF1p, and a second sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase are separated by a linker sequence.

In some embodiments, the linker sequence comprises an internal ribosome entry sequence (IRES). In some embodiments, the IRES is an IRES from CVB3 or EV71. In some embodiments, the linker sequence encodes a self-cleaving peptide sequence. In some embodiments, the linker sequence encodes a T2A, a E2A or a P2A sequence

In some embodiments, the sequence of a human retrotransposon comprises a sequence that encodes ORF1p fused to an additional protein sequence and/or a sequence that encodes ORF2p fused to an additional protein sequence. In some embodiments, the ORF1p and/or the ORF2p is fused to a nuclear retention sequence. In some embodiments, the nuclear retention sequence is an Alu sequence. In some embodiments, the ORF1p and/or the ORF2p is fused to an MS2 coat protein. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises at least one, two, three or more MS2 hairpin sequences. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that promotes or enhances interaction of a poly A tail of the mRNA with the endonuclease and/or a reverse transcriptase. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that promotes or enhances interaction of a poly-A-binding protein (PABP) with the endonuclease and/or a reverse transcriptase. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises a sequence that increases specificity of the endonuclease and/or a reverse transcriptase to the mRNA relative to another mRNA expressed by the cell. In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence comprises an Alu element sequence.

In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase have the same promoter. In some embodiments, the insert sequence has a promoter that is different from the promoter of the first sequence encoding ORF1p. In some embodiments, the insert sequence has a promoter that is different from the promoter of the second sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the first sequence encoding ORF1p and/or the second sequence encoding an endonuclease and/or a reverse transcriptase have a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site and combinations thereof. In some embodiments, the insert sequence has a promoter or transcription initiation site selected from the group consisting of an inducible promoter, a CMV promoter or transcription initiation site, a T7 promoter or transcription initiation site, an EF1a promoter or transcription initiation site and combinations thereof.

In some embodiments, the first sequence encoding ORF1p and the second sequence encoding an endonuclease and/or a reverse transcriptase are codon optimized for expression in a human cell.

In some embodiments, the mRNA comprises a WPRE element. In some embodiments, the mRNA comprises a selection marker. In some embodiments, the mRNA comprises a sequence encoding an affinity tag. In some embodiments, the affinity tag is linked to the sequence encoding an endonuclease and/or a reverse transcriptase.

In some embodiments, the 3′ UTR comprises a poly A sequence or wherein a poly A sequence is added to the mRNA in vitro. In some embodiments, the poly A sequence is downstream of a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the insert sequence is upstream of the poly A sequence.

In some embodiments, the 3′ UTR sequence comprises the insert sequence. In some embodiments, the insert sequence comprises a sequence that is a reverse complement of the sequence encoding the exogenous polypeptide. In some embodiments, the insert sequence comprises a polyadenylation site. In some embodiments, the insert sequence comprises an SV40 polyadenylation site. In some embodiments, the insert sequence comprises a polyadenylation site upstream of the sequence that is a reverse complement of the sequence encoding the exogenous polypeptide. In some embodiments, the insert sequence is integrated into the genome at a locus that is not a ribosomal locus. In some embodiments, the insert sequence integrates into a gene or regulatory region of a gene, thereby disrupting the gene or downregulating expression of the gene. In some embodiments, the insert sequence integrates into a gene or regulatory region of a gene, thereby upregulating expression of the gene. In some embodiments, the insert sequence integrates into the genome and replaces a gene. In some embodiments, the insert sequence is stably integrated into the genome. In some embodiments, the insert sequence is retrotransposed into the genome. In some embodiments, the insert sequence is integrated into the genome by cleavage of a DNA strand of a target site by an endonuclease encoded by the mRNA. In some embodiments, the insert sequence is integrated into the genome via target-primed reverse transcription (TPRT). In some embodiments, the insert sequence is integrated into the genome via reverse splicing of the mRNA into a DNA target site of the genome.

In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the immune cell is a myeloid cell. In some embodiments, the immune cell is selected from a group consisting of a monocyte, a macrophage, a dendritic cell, a dendritic precursor cell, and a macrophage precursor cell.

In some embodiments, the mRNA is a self-integrating mRNA. In some embodiments, the method comprises introducing into the cell the mRNA. In some embodiments, the method comprises introducing into the cell the vector encoding the mRNA. In some embodiments, the method comprises introducing the mRNA or the vector encoding the mRNA into a cell ex vivo. In some embodiments, the method further comprises administering the cell to a human subject. In some embodiments, the method comprises administering the mRNA or the vector encoding the mRNA to a human subject. In some embodiments, an immune response is not elicited in the human subject. In some embodiments, the mRNA or the vector is substantially non-immunogenic.

In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector comprises a non-LTR retrotransposon. In some embodiments, the vector comprises a human L1 element. In some embodiments, the vector comprises a L1 retrotransposon ORF1 gene. In some embodiments, the vector comprises a L1 retrotransposon ORF2 gene. In some embodiments, the vector comprises a L1 retrotransposon.

In some embodiments, the mRNA is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases. In some embodiments, the mRNA is a most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 kilobases.

In some embodiments, the mRNA comprises a payload that is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 kilobases. In some embodiments, the mRNA is a most about 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5 kilobases. In some embodiments, the mRNA is at least about 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6 kilobases. In some embodiments, the mRNA is at least about 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7 kilobases. In some embodiments, the mRNA is at least about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8 kilobases. In some embodiments, the mRNA is at least about 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9 kilobases. In some embodiments, the mRNA is at least about 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 kilobases. In some embodiments, the mRNA is at least about 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9 or 11 kilobases. In some embodiments, the mRNA is at least about 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9 or 12 kilobases. In some embodiments, the mRNA comprises a payload of about 6.8 kB, e.g., a sequence encoding a ABCA4 gene product. In some embodiments, the mRNA comprises a payload of about 6.7 kB, e.g., a sequence encoding a MY07A gene product. In some embodiments, the mRNA comprises a payload of about 7.5 kB, e.g., a sequence encoding a CEP290 gene product. In some embodiments, the mRNA comprises a payload of about 10.1 kB, e.g., a sequence encoding a CDH23 gene product. In some embodiments, the mRNA comprises a payload of about 9.4 kB, e.g., a sequence encoding a EYS gene product. In some embodiments, the mRNA comprises a payload of about 15.6 kB, e.g., a sequence encoding a USH2a gene product. In some embodiments, the mRNA comprises a payload of about 12.5 kB, e.g., a sequence encoding a ALMS1 gene product. In some embodiments, the mRNA comprises a payload of about 4.6 kB, e.g., a sequence encoding a GDE gene product. In some embodiments, the mRNA comprises a payload of about 6 kB, e.g., a sequence encoding the OTOF gene product. In some embodiments, the mRNA comprises a payload of about 7.1 kB, e.g., a sequence encoding a F8 gene product.

One of the advantages of using the method of integration of a nucleic acid into the genome using retrotransposition is that it can be designed as described herein to deliver a nucleic acid cargo that is much larger than that using any other existing methods. For example, lentiviral and adeno-associated viral (AAV) gene delivery method are not expected to deliver a nucleic acid cargo of greater than 4 kB. In addition, lentiviral delivery entails risk of insertional mutagenesis and other toxicities. AAV mediated delivery entails unresolved liver and CNS toxicity. On the other hand, retrotransposition mediated method (Retro-T) using mRNA as described herein is rapid, safer and less complex than these viral methods.

In some embodiments, the mRNA comprises a sequence that inhibits or prevents degradation of the mRNA. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA inhibits or prevents degradation of the mRNA by an exonuclease or an RNAse. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA is a G quadruplex, pseudoknot or triplex sequence. In some embodiments, the sequence the sequence that inhibits or prevents degradation of the mRNA is an exoribonuclease-resistant RNA structure from a flaviviral RNA or an ENE element from KSV. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA inhibits or prevents degradation of the mRNA by a deadenylase. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA comprises non-adenosine nucleotides within or at a terminus of a poly A tail of the mRNA. In some embodiments, the sequence that inhibits or prevents degradation of the mRNA increases stability of the mRNA. In some embodiments, the exogenous sequence comprises a sequence encoding an exogenous polypeptide. In some embodiments, the sequence encoding an exogenous polypeptide is not in frame with a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the sequence encoding an exogenous polypeptide is not in frame with a sequence encoding an endonuclease and/or a reverse transcriptase. In some embodiments, the exogenous sequence does not comprise introns. In some embodiments, the exogenous sequence comprises a sequence encoding an exogenous polypeptide selected from the group consisting of an enzyme, a receptor, a transport protein, a structural protein, a hormone, an antibody, a contractile protein and a storage protein. In some embodiments, the exogenous sequence comprises a sequence encoding an exogenous polypeptide selected from the group consisting of a chimeric antigen receptor (CAR), a ligand, an antibody, a receptor, and an enzyme. In some embodiments, the exogenous sequence comprises a regulatory sequence. In some embodiments, the regulatory sequence comprises a cis-acting regulatory sequence. In some embodiments, the regulatory sequence comprises a cis-acting regulatory sequence selected from the group consisting of an enhancer, a silencer, a promoter or a response element. In some embodiments, the regulatory sequence comprises a trans-acting regulatory sequence. In some embodiments, the regulatory sequence comprises a trans-acting regulatory sequence that encodes a transcription factor.

In some embodiments, integration of the insert sequence does not adversely affect cell health. In some embodiments, the endonuclease, the reverse transcriptase or both are capable of site-specific integration of the insert sequence.

In some embodiments, the mRNA comprises a sequence encoding an additional nuclease domain or a nuclease domain that is not derived from ORF2. In some embodiments, the mRNA comprises a sequence encoding a megaTAL nuclease domain, a TALEN domain, a Cas9 domain, a zinc finger binding domain from an R2 retroelement, or a DNA binding domain that binds to repetitive sequences such as a Rep78 from AAV. In some embodiments, the endonuclease comprises a mutation that reduces activity of the endonuclease compared to the endonuclease without the mutation. In some embodiments, the endonuclease is an ORF2p endonuclease and the mutation is S228P. In some embodiments, the mRNA comprises a sequence encoding a domain that increases fidelity and/or processivity of the reverse transcriptase. In some embodiments, the reverse transcriptase is a reverse transcriptase from a retroelement other than ORF2 or reverse transcriptase that has higher fidelity and/or processivity compared to a reverse transcriptase of ORF2p. In some embodiments, the reverse transcriptase is a group II intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is a group IIA intron reverse transcriptase, a group IIB intron reverse transcriptase, or a group IIC intron reverse transcriptase. In some embodiments, the group II intron reverse transcriptase is TGIRT-II or TGIRT-III.

In some embodiments, the mRNA comprises a sequence comprising an Alu element and/or a ribosome binding aptamer. In some embodiments, the mRNA comprises a sequence encoding a polypeptide comprising a DNA binding domain. In some embodiments, the 3′ UTR sequence is derived from a viral 3′ UTR or a beta-globin 3′ UTR.

In one aspect, provided herein is a composition comprising a recombinant mRNA or vector encoding an mRNA, wherein the mRNA comprises a human LINE-1 transposon sequence comprising a human LINE-1 transposon 5′ UTR sequence, a sequence encoding ORF1p downstream of the human LINE-1 transposon 5′ UTR sequence, an inter-ORF linker sequence downstream of the sequence encoding ORF1p, a sequence encoding ORF2p downstream of the inter-ORF linker sequence, and a 3′ UTR sequence derived from a human LINE-1 transposon downstream of the sequence encoding ORF2p; wherein the 3′ UTR sequence comprises an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element.

In some embodiments, the insert sequence integrates into the genome of a cell when introduced into the cell. In some embodiments, the insert sequence integrates into a gene associated a condition or disease, thereby disrupting the gene or downregulating expression of the gene. In some embodiments, the insert sequence integrates into a gene, thereby upregulating expression of the gene. In some embodiments, the recombinant mRNA or vector encoding the mRNA is isolated or purified.

In one aspect, provided herein is a composition comprising a nucleic acid comprising a nucleotide sequence encoding (a) a long interspersed nuclear element (LINE) polypeptide, wherein the LINE polypeptide includes human ORF1p and human ORF2p; and (b) an insert sequence, wherein the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide or a reverse complement of a sequence encoding an exogenous regulatory element, wherein the composition is substantially non-immunogenic.

In some embodiments, the composition comprises human ORF1p and human ORF2p proteins. In some embodiments, the composition comprises a ribonucleoprotein (RNP) comprising human ORF1p and human ORF2p complexed to the nucleic acid. In some embodiments, the nucleic acid is mRNA.

In one aspect, provided herein is a composition comprising a cell comprising a composition described herein. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell or a B cell. In some embodiments, the immune cell is a myeloid cell. In some embodiments, the immune cell is selected from a group consisting of a monocyte, a macrophage, a dendritic cell, a dendritic precursor cell, and a macrophage precursor cell. In some embodiments, the insert sequence is a reverse complement of a sequence encoding an exogenous polypeptide and the exogenous polypeptide is a chimeric antigen receptor (CAR).

In one aspect, provided herein is a pharmaceutical composition comprising a composition described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is for use in gene therapy. In some embodiments, the pharmaceutical composition is for use in the manufacture of a medicament for treating a disease or condition. In some embodiments, the pharmaceutical composition is for use in treating a disease or condition. In one aspect, provided herein is a method of treating a disease in a subject, comprising administering a pharmaceutical composition described herein to a subject with a disease or condition. In some embodiments, the method increases an amount or activity of a protein or functional RNA in the subject. In some embodiments, the subject has a deficient amount or activity of a protein or functional RNA. In some embodiments, the deficient amount or activity of a protein or functional RNA is associated with or causes the disease or condition.

In some embodiments, the method further comprising administering an agent that inhibits human silencing hub (HUSH) complex, an agent that inhibits FAM208A, or an agent that inhibits TRIM28. In some embodiments, the agent that inhibits human silencing hub (HUSH) complex is an agent that inhibits Periphilin, TASOR and/or MPP8. In some embodiments, the agent that inhibits human silencing hub (HUSH) complex inhibits assembly of the HUSH complex.

In some embodiments, the agent inhibits the fanconia anemia complex. In some embodiments, the agent inhibits FANCD2-FANC1 heterodimer monoubiquitination. In some embodiments, the agent inhibits FANCD2-FANC1 heterodimer formation. In some embodiments the agent inhibits the Fanconi Anemia (FA) core complex. FA core complex is a component of the fanconi anemia DNA damage repair pathway, e.g., in chemotherapy induced DNA inter-strand crosslinks. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Examples of agents that can inhibit the FA core complex include Bortezomib and curcumin analogs EF24 and 4H-TTD.

In some embodiments, the sequences to be inserted may be placed under the control of tissue-specific elements, such that the entire inserted DNA is only functional in those cells in which the tissue-specific element is active.

In one aspect, provided herein are method and compositions for stable gene transfer to a cell by introducing to the cell a heterologous nucleic acid or gene of interest (e.g., a transgene, a regulatory sequence, for example, a sequence for an inhibitory nucleic acid, an siRNA, a miRNA), flanked by sequences that cause retrotransposition of the heterologous nucleic acid sequence into the genome of the cell. In some embodiments, the heterologous nucleic acid is termed insert for the purpose of the description in this document, where the insert is the nucleic acid sequence that will be reverse transcribed and inserted into the genome of the cell by the intended design of the constructs described herein. In some embodiments, the heterologous nucleic acid is also termed the cargo, or cargo sequence for the purpose of the description in this document. The cargo can comprise the sequence of the heterologous nucleic acid that that is inserted in the genome. In some embodiments, the cell may be a cell mammalian cell. The mammalian cell may be of epithelial, mesothelial or endothelial origin. In some embodiments, the cell may be a stem cell. In some embodiments, the cell may be a precursor cell. In some embodiments, the cell may be a cell that is terminally differentiated. In some embodiments, the cell may be a muscle cell, a cardiac cell, an epithelial cell, a hematopoietic cell, a mucous cell, an epidermal cell, a squamous cell, a cartilage cell, a bone cell, or any cell of mammalian origin. In some embodiments, the cell is of hematopoietic lineage. In some embodiments, the cell is of myeloid lineage, or a phagocytic cell, for example a monocyte, macrophage, a dendritic cell or a myeloid precursor cell. In some embodiments, the nucleic acid encoding the transgene is an mRNA.

In some embodiments, the retrotransposable elements may be derived from a non-LTR retrotransposon.

Provided herein is a method of integrating a nucleic acid sequence into a genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA into the cell, wherein the mRNA comprises an insert sequence and wherein the insert sequence is integrated into the genome of the cell. In some embodiments, the insert sequence comprises (i) an exogenous sequence, or (ii) a sequence that is a reverse complement of the exogenous sequence; a 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for a human ORF protein. In some embodiments, the ORF protein is a human LINE 1 ORF2 protein. In some embodiments, the ORF protein is a non-human ORF protein. In some embodiments, the ORF protein is a chimeric protein, a recombinant protein or an engineered protein.

Provided herein is a method for integrating a nucleic acid sequence into the genome of an immune cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises, (a) an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; (b) 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence, wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and a reverse transcriptase binding site, and wherein the transgene sequence is integrated into the genome of the immune cell.

In some embodiments, the structural elements that mediate RNA integration or transposition may be encoded in a synthetic construct and are relied upon to deliver a heterologous gene of interest to the cell. In some embodiments, the synthetic construct may comprise a nucleic acid encoding the heterologous gene of interest and the structural elements that cause integration or retrotransposition of a heterologous gene of interest into the genome. In some embodiments, the structural elements that cause integration or retrotransposition may include a 5′ L1 RNA region, and a 3′-L1 region, the latter comprising a poly A 3′ region for priming. In some embodiments, the 5′ L1 RNA region may comprise one or more stem loop regions. In some embodiments, the L1-3′ region may comprise one or more stem loop regions. In some embodiments, the 5′- and 3′ L1 regions are constructed as flanking the nucleic acid sequence encoding the heterologous gene of interest (the transgene). In some embodiments, the structural elements may include a region from an L1 or an Alu RNA comprising the hairpin loop structure that includes the A-Box and the B-Box elements that are ribosomal binding sites In some embodiments, the synthetic nucleic acid may comprise a L1-Ta promoter.

There may be two types of LINE RNA recognition by ORF2p—the stringent and the relaxed. In the stringent type RT recognizes its own 3′UTR tail, and in the relaxed type RT does not require any specific recognition except for the poly-A tail. Division into the stringent and the relaxed type came from the observation that some LINE/SINE pairs share the same 3′-end. For the stringent type, the experimental studies showed that a 3′UTR stem-loop promotes retrotransposition. The 5′-UTR of the LINE retrotransposition sequences have been shown to contain three conserved stem loop regions.

In some embodiments, the transgene, or transcript of interest may be flanked by transposable elements from a L1 or an Alu sequence at the 5′ and the 3′ end. In some embodiments, the 5′ region of a retrotransposon comprises an Alu sequence. In some embodiments, the 3′ region of a retrotransposon comprises an Alu sequence. In some embodiments, the 5′ region of a retrotransposon comprises an L1 sequence. In some embodiments, the 3′ region of a retrotransposon comprises an L1 sequence. In some embodiments, the transgene or transcript of interest is flanked by an SVA transposon sequence.

In some embodiments, the transcript of interest may comprise an L1 or an Alu sequence, encoding the binding regions for ORF2p and the 3′-poly A priming regions. In some embodiments, the heterologous nucleic acid encoding the transgene of interest may be flanked by an L1 or an Alu sequence, encoding the binding regions for ORF1p and the 3′-poly A priming regions. The 3′-region may comprise one or more stem loop structures. In some embodiments, the transcript of interest is structured for cis integration or retrotransposition. In some embodiments, the transcript of interest is structured for trans integration or retrotransposition.

In some embodiments, the retrotransposon is a human retrotransposon. The sequence of a human retrotransposon can comprise a sequence encoding an endonuclease and/or a reverse transcriptase. The sequence of a human retrotransposon can encode for two proteins that are translated from a single RNA containing two non-overlapping ORFs. In some embodiments, the two ORFs are ORF1 and ORF2.

Accordingly, provided herein is a method for stably integrating a heterologous nucleic acid encoding a transgene into the genome of a cell, such as a myeloid cell, the method comprising introducing to the cell a nucleic acid encoding: the transgene; one or more 5′ nucleic acid sequences flanking the region encoding the transgene, comprising a 5′ region of a retrotransposon; and one or more 3′ nucleic acid sequence flanking the region encoding the transgene, comprising a 3′ region of a retrotransposon, wherein the 3′ region of the retrotransposon comprises a genomic DNA priming sequence and a LINE transposase binding sequence, having the respective endonuclease and reverse transcriptase (RT) activity.

Provided herein is a method for integrating a nucleic acid sequence into the genome of a cell, the method comprising introducing a recombinant mRNA or a vector encoding an mRNA, wherein the mRNA comprises an insert sequence, wherein the insert sequence comprises (i) an exogenous sequence or (ii) a sequence that is a reverse complement of the exogenous sequence; (b) a 5′ UTR sequence, a sequence of a human retrotransposon downstream of the 5′ UTR sequence, and a 3′ UTR sequence downstream of the sequence of a human retrotransposon; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises an endonuclease binding site and a reverse transcriptase binding site, and wherein the sequence of a human retrotransposon encodes for two proteins that are translated from a single RNA containing two ORFs, and wherein the insert sequence is integrated into the genome of the cell.

In some embodiments, the method comprising using a single nucleic acid molecule for delivering and integrating the insert sequence into the genome of a cell. The single nucleic acid molecule may be a plasmid vector. The single nucleic acid may be DNA or an RNA molecule. The single nucleic acid may be an mRNA.

In some embodiments, the method comprises introducing into a cell one or more polynucleotides comprising the human retrotransposon and a heterologous nucleic acid sequence. In some embodiments, the one or more polynucleotides comprises (i) a first nucleic acid molecule encoding an ORF1p; (ii) a second nucleic acid molecule encoding an ORF2p and a sequence encoding a cargo. In some embodiments, the first nucleic acid and the second nucleic acid are mRNA. In some embodiments, the first nucleic acid and the second nucleic acid are DNA, e.g., encoded in separate plasmid vectors.

Provided herein is a self-integrating polynucleotide that comprises a sequence which is inserted into the genome of a cell, and insert is stably integrated into the genome by the self-integrating naked polynucleotide. In some embodiments, the polynucleotide is an RNA. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is an mRNA that has modifications. In some embodiments, the modifications ensure protection against RNases in the intracellular milieu. In some embodiments, the modifications include substituted modified nucleotides, e.g., 5-methylcytidine, pseudouridine or 2-thiouridine.

In some embodiments, a single polynucleotide is used for delivery and genomic integration of the insert (or cargo) nucleic acid. In some embodiments, the single polynucleotide is bicistronic. In some embodiments, the single polynucleotide is tricistronic. In some embodiments, the single polynucleotide is multi-cistronic. In some embodiments, a two or more polynucleotide molecules are used for delivery and genomic integration of the insert (or cargo) nucleic acid.

In some embodiments, a retrotransposable genetic element may be generated, the retrotransposable genetic element comprising (i) a heterologous nucleic acid encoding a transgene or a non-coding sequence to be inserted into the genome of a cell (the insert); (ii) a nucleic sequence encoding one or more retrotransposon ORF-encoding sequences; (iii) one or more UTR regions of the ORF-coding sequences, such that the heterologous nucleic acid encoding a transgene or a non-coding sequence to be inserted is comprised within the UTR sequences; wherein the 3′ region of the retrotransposon ORF-encoding sequences comprises a genomic DNA priming sequence.

In some embodiments, the retrotransposable genetic element may be introduced into a cell for stably integrating the transgene into the genomic DNA. In some embodiments, the retrotransposable genetic element comprises (a) a retrotransposon protein coding sequence, and a 3′ UTR; and (b) a sequence comprising a heterologous nucleic acid that is to be inserted (e.g, integrated) within the genome of a cell. The retrotransposon protein coding sequence, and the 3′ UTR may be a complete and sufficient unit for delivering the heterologous nucleic acid sequence within the genome of the cell, and comprise the retrotransposable elements, such as an endonuclease, a reverse transcriptase, a sequence in the 3′ UTR for binding to and priming the genomic DNA at the region cleaved by the endonuclease to start reverse transcribing and incorporating the heterologous nucleic acid.

In some embodiments, the coding sequence of the insert is in forward orientation with respect to the coding sequence of the one or more ORFs. In some embodiments, the coding sequence of the insert is in reverse orientation with respect to the coding sequence of the one or more ORFs. The coding sequence of the insert and the coding sequence of the one or more ORFs may comprise distinct regulatory elements, including 5′ UTR, 3′ UTR, promoter, enhancer, etc. In some embodiments, the 3′ UTR or the 5′-UTR of the insert may comprise the coding sequence of the one or more ORFs, and likewise, the coding sequence of the insert may be situated within in the 3′ UTR of the coding sequence of the one or more ORFs.

In some embodiments, a retrotransposable genetic element may be generated, the retrotransposable genetic element comprising: (a) an insert sequence, comprising (i) an exogenous sequence, a sequence that is a reverse complement of the exogenous sequence; a 5′ UTR sequence and a 3′ UTR sequence downstream of the 5′ UTR sequence; wherein the 5′ UTR sequence or the 3′ UTR sequence comprises a binding site for a human ORF protein.

In some embodiments, the retrotransposon may comprise a SINE or LINE element. In some embodiments, the retrotransposon comprises a SINE or LINE stem loop structure, such as an Alu element.

In some embodiments, the retrotransposon is a LINE-1 (L1) retrotransposon. In some embodiments, the retrotransposon is human LINE-1. Human LINE-1 sequences are abundant in the human genome. There are approximately 13,224 total human L1-s, of which 480 are active, which make up about 3.6%. Therefore, human L1 proteins are well tolerated and non-immunogenic in humans. Moreover, a tight regulation of random transposition in human ensures that random transposase activity will not be triggered by introduction of the L1 system as described herein. In addition, the retrotransposable constructs designed herein may comprise targeted and specific incorporation of the insert sequence. In some embodiments, the retrotransposable genetic element may comprise designs intended to overcome the silencing machinery actively prevalent in human cells, while being careful that random integration resulting in genomic instability is not initiated.

Accordingly, the retrotransposable constructs may comprise a sequence encoding a human LINE-1 ORF1 protein; and a human LINE-1 ORF2 protein. In some embodiments, the construct comprises a nucleic acid sequence encoding an ORF1p protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to MGKKQNRKTGNSKTQSASPPPKERSSSPATEQSWMENDFDELREEGFRRSNYSELREDIQTKGK EVENFEKNLEECITRITNTEKCLKELMELKTKARELREECRSLRSRCDQLEERVSAMEDEMNEM KREGKFREKRIKRNEQSLQEIWDYVKRPNLRLIGVPESDVENGTKLENTLQDIIQENFPNLARQA NVQIQEIQRTPQRYSSRRATPRHIIVRFTKVEMKEKMLRAAREKGRVTLKGKPIRLTVDLSAETL QARREWGPIFNILKEKNFQPRISYPAKLSFISEGEIKYFIDKQMLRDFVTTRPALKELLKEALNME RNNRYQPLQNHAKM (SEQ ID NO: 53). In some embodiments, the construct comprises a nucleic acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to

(SEQ ID NO: 54) atgggcaagaagcaaaatcgcaagacggggaattccaagacacaatccg ctagcccaccacctaaagagcgttctagctcccctgctactgagcagtc ctggatggaaaacgacttcgatgaactccgggaagagggatttaggcga tccaactattcagaactccgcgaagatatccagacaaaggggaaggaag tcgagaatttcgagaagaacctcgaggagtgcatcacccgtatcacaaa cactgagaaatgtctcaaagaactcatggaacttaagacaaaagccagg gagcttcgagaggagtgtcggagtctgagatccaggtgtgaccagctcg aggagcgcgtgagcgcgatggaagacgagatgaacgagatgaaaagaga gggcaaattcagggagaagcgcattaagaggaacgaacagagtctgcag gagatttgggattacgtcaagaggcctaacctgcggttgatcggcgtcc ccgagagcgacgtagaaaacgggactaaactggagaatacacttcaaga catcattcaagaaaattttccaaacctggctcggcaagctaatgtgcaa atccaagagatccaacgcacaccccagcggtatagctctcggcgtgcca cccctaggcatattatcgtgcgctttactaaggtggagatgaaagagaa gatgctgcgagccgctcgggaaaagggaagggtgactttgaagggcaaa cctattcggctgacggttgaccttagcgccgagacactccaggcacgcc gggaatggggccccatctttaatatcctgaaggagaagaacttccagcc acgaatctcttaccctgcaaagttgagttttatctccgagggtgagatt aagtatttcatcgataaacagatgctgcgagacttcgtgacaactcgcc cagctctcaaggaactgctcaaagaggctcttaatatggagcgcaataa tagatatcaacccttgcagaaccacgcaaagatgtga.

In some embodiments, the construct comprises a nucleic acid sequence encoding an ORF2p protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to MTGSNSHITILTLNINGLNSAIKRHRLASWIKSQDPSVCCIQETHLTCRDTHRLKIKGWRKIYQAN GKQKKAGVAILVSDKTDFKPTKIKRDKEGHYIMVKGSIQQEELTILNIYAPNTGAPRFIKQVLSDL QRDLDSHTLIMGDFNTPLSTLDRSTRQKVNKDTQELNSALHQADLIDIYRTLHPKSTEYTFFSAP HHTYSKIDHIVGSKALLSKCKRTEIITNYLSDHSAIKLELRIKNLTQSRSTTWKLNNLLLNDYWV HNEMKAEIKMFFETNENKDTTYQNLWDAFKAVCRGKFIALNAYKRKQERSKIDTLTSQLKELE KQEQTHSKASRRQEITKIRAELKEIETQKTLQKINESRSWFFERINKIDRPLARLIKKKREKNQIDTI KNDKGDITTDPTEIQTTIREYYKHLYANKLENLEEMDTFLDTYTLPRLNQEEVESLNRPITGSEIV AIINSLPTKKSPGPDGFTAEFYQRYMEELVPFLLKLFQSIEKEGILPNSFYEASIILIPKPGRDTTKKE NFRPISLMNIDAKILNKILANRIQQHIKKLIHHDQVGFIPGMQGWFNIRKSINVIQHINRAKDKNH MIISIDAEKAFDKIQQPFMLKTLNKLGIDGTYFKIIRAIYDKPTANIILNGQKLEAFPLKTGTRQGC PLSPLLFNIVLEVLARAIRQEKEIKGIQLGKEEVKLSLFADDMIVYLENPIVSAQNLLKLISNFSKV SGYKINVQKSQAFLYTNNRQTESQIMGELPFVIASKRIKYLGIQLTRDVKDLFKENYKPLLKEIKE DTNKWKNIPCSWVGRINIVKMAILPKVIYRFNAIPIKLPMTFFTELEKTTLKFIWNQKRARIAKSIL SQKNKAGGITLPDFKLYYKATVTKTAWYWYQNRDIDQWNRTEPSEIMPHIYNYLIFDKPEKNK QWGKDSLFNKWCWENWLAICRKLKLDPFLTPYTKINSRWIKDLNVKPKTIKTLEENLGITIQDIG VGKDFMSKTPKAMATKDKIDKWDLIKLKSFCTAKETTIRVNRQPTTWEKIFATYSSDKGLISRIY NELKQIYKKKTNNPIKKWAKDMNRHFSKEDIYAAKKHMKKCSSSLAIREMQIKTTMRYHLTPV RMAIIKKSGNNRCWRGCGEIGTLLHCWWDCKLVQPLWKSVWRFLRDLELEIPFDPAIPLLGIYP NEYKSCCYKDTCTRMFIAALFTIAKTWNQPKCPTMIDWIKKMWHIYTMEYYAAIKNDEFISFVG TWMKLETIILSKLSQEQKTKHRIFSLIGGN (SEQ ID NO: 55). In some embodiments, the construct comprises a nucleic acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to

(SEQ ID NO: 56) atgaccggctctaactcacatatcaccatccttacacttaacattaacggcctcaactcagctatcaagcgccatcggctggccagctggatcaa atcacaggatccaagcgtttgttgcatccaagagacccacctgacctgtagagatactcaccgcctcaagatcaagggatggcgaaagatttatc aggcgaacggtaagcagaagaaagccggagtcgcaattctggtctcagacaagacggatttcaagcccaccaaaattaagcgtgataaggaaggt cactatattatggtgaaaggcagcatacagcaggaagaacttaccatattgaacatctacgcgccaaacaccggcgcacctcgctttatcaaaca ggtcctgtccgatctgcagcgagatctggattctcatacgttgattatgggtgatttcaatacaccattgagcaccctggatcgcagcaccaggc aaaaggtaaataaagacacgcaagagctcaatagcgcactgcatcaggcagatctcattgatatttatcgcactcttcatcctaagagtaccgag tacacattcttcagcgccccacatcatacatactcaaagatcgatcatatcgtcggctcaaaggctctgctgtcaaagtgcaagcgcacagagat aattacaaattacctgtcagatcatagcgcgatcaagctcgagctgagaatcaagaacctgacccagagccggagtaccacttggaagcttaata acctgctgctcaacgattattgggtccacaatgagatgaaggcagagattaaaatgttcttcgaaacaaatgagaataaggatactacctatcaa aacctttgggatgcctttaaggccgtctgcagaggcaagttcatcgccctcaacgcctataaaagaaaacaagagagatctaagatcgatactct cacctctcagctgaaggagttggagaaacaggaacagacccactccaaggcgtcaagacggcaggagatcacaaagattcgcgccgagttgaaag agatcgaaacccaaaagactcttcagaaaattaacgagtctcgtagttggttcttcgagcggattaataagatagacagacctctggcacgactg attaagaagaagcgcgaaaagaaccagattgataccatcaagaacgacaagggcgacatcactactgacccgaccgagatccagaccactattcg ggagtattataagcatttgtatgctaacaagcttgagaacctggaagagatggacacttttctggatacctatactctgccacggcttaatcaag aggaagtcgagtccctcaaccgcccaattacaggaagcgagattgtggccataattaactccctgccgacaaagaaatctcctggtccggacggg tttacagctgagttttatcaacggtatatggaagagcttgtaccgtttctgctcaagctctttcagtctatagaaaaggaaggcatcttgcccaa ttccttctacgaagcttctataatacttattcccaaaccaggacgcgataccacaaagaaggaaaacttccggcccattagtctcatgaatatcg acgctaaaatattgaacaagattctcgccaacagaatccaacaacatattaagaaattgatacatcacgaccaggtggggtttatacctggcatg cagggctggtttaacatccggaagagtattaacgtcattcaacacattaatagagctaaggataagaatcatatgatcatctctatagacgcgga aaaggcattcgataagattcagcagccatttatgctcaagactctgaacaaactcggcatcgacggaacatattttaagattattcgcgcaattt acgataagccgactgctaacattatccttaacggccaaaagctcgaggcctttccgctcaagactggaacccgccaaggctgtcccctctccccg cttttgtttaatattgtactcgaggtgctggctagggctattcgtcaagagaaagagattaaagggatacagctcgggaaggaagaggtcaagct ttccttgttcgccgatgatatgattgtgtacctggagaatcctattgtgtctgctcagaaccttcttaaacttatttctaactttagcaaggtca gcggctataagattaacgtccagaaatctcaggcctttctgtacacaaataatcgacagaccgaatcccagataatgggtgagcttccgtttgtc atagccagcaaaaggataaagtatctcggaatccagctgacacgagacgttaaagatttgtttaaggaaaattacaagcctctcctgaaagagat taaggaagatactaataagtggaagaatatcccctgttcatgggttggcagaatcaacatagtgaagatggcaatacttcctaaagtgatatatc gctttaacgccatcccaattaaactgcctatgaccttctttacggagctcgagaaaacaacccttaaatttatatggaatcaaaagagagcaaga atagcgaagtccatcttgagccagaagaataaggccggtgggattactttgcctgattttaagttgtattataaagccacagtaactaagacagc ctggtattggtatcagaatagagacatcgaccagtggaatcggaccgaaccatcagagataatgccccacatctataattaccttatattcgata agccagaaaagaataaacagtggggcaaagacagcctcttcaacaagtggtgttgggagaattggctggccatatgccggaaactcaagctcgac ccctttcttacaccctacactaaaatcaacagtaggtggatcaaggacttgaatgtcaagccaaagactataaagacactggaagagaatcttgg gatcacaatacaagatataggcgtcggcaaagattttatgtcaaagacgcccaaggccatggccactaaggataagattgataagtgggacctta ttaagctcaaaagcttctgtactgccaaggagaccacgatcagagttaataggcagcccactacatgggaaaagattttcgccacttattcatca gataaggggttgataagcagaatatataacgagctgaagcagatctacaagaagaaaacgaataatcccatcaagaagtgggcaaaagatatgaa caggcattttagcaaagaggatatctacgccgcgaagaagcatatgaagaagtgtagttcaagcttggccattcgtgagatgcagattaagacga ccatgcgataccaccttaccccagtgaggatggcaattatcaagaaatctggcaataatagatgttggcggggctgtggcgagattggcaccctg ctccattgctggtgggattgcaagctggtgcagccgctttggaaatcagtctggcgctttctgagggacctcgagcttgagattcccttcgatcc cgcaattcccttgctcggaatctatcctaacgaatacaagagctgttgttacaaggatacgtgtacccggatgttcatcgcggccttgtttacga tagctaagacgtggaatcagcctaagtgccccacaatgatcgattggatcaagaaaatgtggcatatttataccatggagtattacgcagcaatt aagaatgacgaatttatttccttcgttgggacctggatgaagctggagactattattctgagcaagctgtctcaggagcaaaagacaaagcatag aatcttctctctcattggtggtaactaa.

In some embodiments, the construct comprises a nucleic acid sequence encoding an ORF2p protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to MVIGTYISIITLNVNGLNAPTKRHRLAEWIQKQDPYICCLQETHFRPRDTYRLKVRGWKKIFHAN GNQKKAGVAILISDKIDFKIKNVTRDKEGHYIMIQGSIQEEDITIINIYAPNIGAPQYIRQLLTAIKE EIDSNTIIVGDFNTSLTPMDRSSKMKINKETEALNDTIDQIDLIDIYRTFHPKTADYTFFSSAHGTFS RIDHILGHKSSLSKFKKIEIISSIFSDHNAMRLEMNHREKNVKKTNTWRLNNTLLNNQEITEEIKQ EIKKYLETNDNENTTTQNLWDAAKAVLRGKFIAIQAYLKKQEKSQVNNLTLHLKKLEKEEQTK PKVSRRKEIIKIRAEINEIETKKTIAKINKTKSWFFEKINKIDKPLARLIKKKRERTQINKIRNEKGE VTTDTAEIQNILRDYYKQLYANKMDNLEEMDKFLERYNLPRLNQEETENINRPITSNEIETVIKNL PTNKSPGPDGFTGEFYQTFREELTPILLKLFQKIAEEGTLPNSFYEATITLIPKPDKDTTKKENYRPI SLMNIDAKILNKILANRIQQHIKRIIHHDQVGFIPGMQGFFNIRKSINVIHHINKLKKKNHMIISIDA EKAFDKIQHPFMIKTLQKVGIEGTYLNIIKAIYDKPTANIILNGEKLKAFPLRSGTRQGCPLSPLLF NIVLEVLATAIREEKEIKGIQIGKEEVKLSLFADDMILYIENPKTATRKLLELINEYGKVAGYKINA QKSLAFLYTNDEKSEREIMETLPFTIATKRIKYLGINLPKETKDLYAENYKTLMKEIKDDTNRWR DIPCSWIGRINIVKMSILPKAIYRFNAIPIKLPMAFFTELEQIILKFVWRHKRPRIAKAVLRQKNGA GGIRLPDFRLYYKATVIKTIWYWHKNRNIDQWNKIESPEINPRTYGQLIYDKGGKDIQWRKDSLF NKWCWENWTATCKRMKLEYSLTPYTKINSKWIRDLNIRLDTIKLLEENIGRTLFDINHSKIFFDPP PRVMEIKTKINKWDLMKLQSFCTAKETINKTKRQPSEWEKIFANESTDKGLISKIYKQLIQLNIKE TNTPIQKWAEDLNRHFSKEDIQTATKHMKRCSTSLIIREMQIKTTMRYHLTPVRMGIIRKSTNNK CWRGCGEKGTLLHCWWECKLIQPLWRTIWRFLKKLKIELPYDPAIPLLGIYPEKTVIQKDTCTR MFIAALFTIARSWKQPKCPSTDEWIKKMWYIYTMEYYSAIKRNEIGSFLETWMDLETVIQSEVSQ KEKNKYRILTHICGTWKNGTDEPVCRTEIETQM (SEQ ID NO: 57). In some embodiments, the construct comprises a nucleic acid sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to

(SEQ ID NO: 58) atggtcataggaacatacatatcgataattaccttaaacgtgaatggattaaatgccccaaccaaaagacatagactggctgaatggatacaaaa acaagacccatatatatgctgtctacaagagacccacttcagacctagggacacatacagactgaaagtgaggggatggaaaaagatattccatg caaatggaaatcaaaagaaagctggagtagctatactcatatcagataaaatagactttaaaataaagaatgttacaagagacaaggaaggacac tacataatgatccagggatcaatccaagaagaagatataacaattataaatatatatgcacccaacataggagcacctcaatacataaggcaact gctaacagctataaaagaggaaatcgacagtaacacaataatagtgggggactttaacacctcacttacaccaatggacagatcatccaaaatga aaataaataaggaaacagaagctttaaatgacacaatagaccagatagatttaattgatatatataggacattccatccaaaaacagcagattac acgttcttctcaagtgcgcacggaacattctccaggatagatcacatcttgggtcacaaatcaagcctcagtaaatttaagaaaattgaaatcat atcaagcatcttttctgaccacaacgctatgagattagaaatgaatcacagggaaaaaaacgtaaaaaagacaaacacatggaggctaaacaata cgttactaaataaccaagagatcactgaagaaatcaaacaggaaataaaaaaatacctagagacaaatgacaatgaaaacacgacgacccaaaac ctatgggatgcagcaaaagcggttctaagagggaagtttatagctatacaagcctacctaaagaaacaagaaaaatctcaagtaaacaatctaac cttacacctaaagaaactagagaaagaagaacaaacaaaacccaaagttagcagaaggaaagaaatcataaagatcagagcagaaataaatgaaa tagaaacaaagaaaacaatagcaaagatcaataaaactaaaagttggttctttgagaagataaacaaaattgataagccattagccagactcatc aagaaaaagagggagaggactcaaatcaataaaatcagaaatgaaaaaggagaagttacaacagacaccgcagaaatacaaaacatcctaagaga ctactacaagcaactttatgccaataaaatggacaacctggaagaaatggacaaattcttagaaaggtataaccttccaagactgaaccaggaag aaacagaaaatatcaacagaccaatcacaagtaatgaaattgaaactgtgattaaaaatcttccaacaaacaaaagtccaggaccagatggcttc acaggtgaattctatcaaacatttagagaagagctaacacccatccttctcaaactcttccaaaaaattgcagaagaaggaacactcccaaactc attctatgaggccaccatcaccctgataccaaaaccagacaaagacactacaaaaaaagaaaattacagaccaatatcactgatgaatatagatg caaaaatcctcaacaaaatactagcaaacagaatccaacaacacattaaaaggatcatacaccacgatcaagtgggatttatcccagggatgcaa ggattcttcaatatacgcaaatcaatcaatgtgatacaccatattaacaaattgaagaagaaaaaccatatgatcatctcaatagatgcagaaaa agcttttgacaaaattcaacacccatttatgataaaaactctccagaaagtgggcatagagggaacctacctcaacataataaaggccatatatg acaaacccacagcaaacatcattctcaatggtgaaaaactgaaagcatttcctctaagatcaggaacgagacaaggatgtccactctcaccacta ttattcaacatagttctggaagtcctagccacggcaatcagagaagaaaaagaaataaaaggaatacaaattggaaaagaagaagtaaaactgtc actgtttgcggatgacatgatactatacatagagaatcctaaaactgccaccagaaaactgctagagctaattaatgaatatggtaaagttgcag gttacaaaattaatgcacagaaatctcttgcattcctatacactaatgatgaaaaatctgaaagagaaattatggaaacactcccatttaccatt gcaacaaaaagaataaaatacctaggaataaacctacctaaggagacaaaagacctgtatgcagaaaactataagacactgatgaaagaaattaa agatgataccaacagatggagagatataccatgttcttggattggaagaatcaacattgtgaaaatgagtatactacccaaagcaatctacagat tcaatgcaatccctatcaaattaccaatggcattttttacggagctagaacaaatcatcttaaaatttgtatggagacacaaaagaccccgaata gccaaagcagtcttgaggcaaaaaaatggagctggaggaatcagactccctgacttcagactatactacaaagctacagtaatcaagacaatatg gtactggcacaaaaacagaaacatagatcaatggaacaagatagaaagcccagagattaacccacgcacctatggtcaactaatctatgacaaag gaggcaaagatatacaatggagaaaagacagtctcttcaataagtggtgctgggaaaactggacagccacatgtaaaagaatgaaattagaatac tccctaacaccatacacaaaaataaactcaaaatggattagagacctaaatataagactggacactataaaactcttagaggaaaacataggaag aacactctttgacataaatcacagcaagatctttttcgatccacctcctagagtaatggaaataaaaacaaaaataaacaagtgggacctaatga aacttcaaagcttttgcacagcaaaggaaaccataaacaagacgaaaagacaaccctcagaatgggagaaaatatttgcaaatgaatcaacggac aaaggattaatctccaaaatatataaacagctcattcagctcaatatcaaagaaacaaacaccccaatccaaaaatgggcagaagacctaaatag acatttctccaaagaagacatacagacggccacgaagcacatgaaaagatgctcaacatcactaattattagagaaatgcaaatcaaaactacaa tgaggtatcacctcactcctgttagaatgggcatcatcagaaaatctacaaacaacaaatgctggagagggtgtggagaaaagggaaccctcttg cactgttggtgggaatgtaaattgatacagccactatggagaacaatatggaggttccttaaaaaactaaaaatagaattaccatatgacccagc aatcccactactgggcatatacccagagaaaaccgtaattcaaaaagacacatgcacccgaatgttcattgcagcactatttacaatagccaggt catggaagcaacctaaatgcccatcgacagacgaatggataaagaagatgtggtacatatatacaatggaatattactcagccataaaaaggaac gaaattgggtcatttttagagacgtggatggatctagagactgtcatacagagtgaagtaagtcagaaagagaaaaacaaatatcgtatattaac gcatatatgtggaacctggaaaaatggtacagatgaaccggtctgcaggacagaaattgagacacaaatgtaa.

In some embodiments, the construct comprises a nucleic acid sequence encoding a nuclear localization sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to PAAKRVKLD ((SEQ ID NO: 59). In some embodiments, the nuclear localization sequence is fused to the ORF2p sequence. In some embodiments, the construct comprises a nucleic acid sequence encoding a flag tag having the sequence DYKDDDDK (SEQ ID NO: 60). In some embodiments, the flag tag is fused to the ORF2p sequence. In some embodiments, the flag tag is fused to the nuclear localization sequence.

In some embodiments, the construct comprises a nucleic acid sequence encoding an MS2 coat protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to ASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEV PKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYAMASNFTQFVLVD NGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLN MELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 61). In some embodiments, the MS2 coat protein sequence is fused to the ORF2p sequence.

In some embodiments, the transgene may comprise a flanking sequence which comprises an Alu ORF2p recognition sequence.

In some embodiments, additional elements may be introduced into the mRNA. In some embodiments, the additional elements may be an IRES element or a T2A element. In some embodiments, the mRNA transcript comprises one, two, three or more stop codons at the 3′-end.

In some embodiments, the one, two, three or more stop codons are designed to be in tandem. In some embodiments, the one, two, three or more stop codons are designed to be in all three reading frames. In some embodiments, the one, two, three or more stop codons may be designed to be both in multiple reading frames and in tandem.

In some embodiments, one or more target specific nucleotides may be added at the priming end of the L1 or the Alu RNA priming region.

In some embodiments, the 5′ UTR sequence or the 3′ UTR sequence in addition to be able to bind the ORF protein may also be capable of binding to one or more endogenous proteins that regulate gene retrotransposition and/or stable integration. In some embodiments, the flanking sequence is capable of binding to a PABP protein.

In some embodiments, the 5′ region flanking the transcript may comprise a strong promoter. In some embodiments, the promoter is a CMV promoter.

In some embodiments, an additional nucleic encoding L1 ORF2p is introduced into the cell. In some embodiments, the sequence encoding L1 ORF 1 is omitted, and only L1-ORF2 is included. In some embodiments, the nucleic acid encoding the transgene with the flanking elements is mRNA. In some embodiments, the endogenous L1-ORF1p function may be suppressed or inhibited.

In some embodiments, the nucleic acid encoding the transgene with the retrotransposition flanking elements comprise one or more nucleic acid modifications. In some embodiments, the nucleic acid encoding the transgene with the retrotransposition flanking elements comprises one or more nucleic acid modifications in the transgene. In some embodiments, the modifications comprise codon optimization of the transgene sequence. In some embodiments, the codon optimization is for more efficient recognition by the human translational machinery, leading to more efficient expression in a human cell. In some embodiments, the one or more nucleic acid modification is performed in the 5′-flanking sequence or the 3′-flanking sequence including one or more stem-loop regions. the nucleic acid encoding the transgene with the retrotransposition flanking elements comprise one, two, three, four, five, six, seven eight, nine, ten or more nucleic acid modifications.

In some embodiments, the retrotransposed transgene is stably expressed for the life of the cell. In some embodiments, the cell is a myeloid cell. In some embodiments, the myeloid cell is a monocyte precursor cell. In some embodiments, the myeloid cell is an immature monocyte. In some embodiments, the monocyte is an undifferentiated monocyte. In some embodiments, the myeloid cell is a CD14+ cell. In some embodiments, the myeloid cell does not express CD16 marker. In some embodiments, the myeloid cell is capable of remaining functionally active for a desired period of greater than 3 days, greater than 4 days, greater than 5 days, greater than 6 days, greater than 7 days, greater than 8 days, greater than 9 days, greater than 10 days, greater than 11 days, greater than 12 days, greater than 13 days, greater than 14 days or more under suitable conditions. A suitable condition may denote an in vitro condition, or an in vivo condition or a combination of both.

In some embodiments, the retrotransposed transgene may be stably expressed in the cell for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days. In some embodiments, the retrotransposed transgene is stably expressed in the cell for more than 10 days. In some embodiments, the retrotransposed transgene is stably expressed in the cell for more than 2 weeks. In some embodiments, the retrotransposed transgene is stably expressed in the cell for about 1 month.

In some embodiments, the retrotransposed transgene may be modified for stable expression. In some embodiments, the retrotransposed transgene may be modified for resistant to in vivo silencing.

In some embodiments, the expression of the retrotransposed transgene may be controlled by a strong promoter. In some embodiments, the expression of the retrotransposed transgene may be controlled by a moderately strong promoter. In some embodiments, the expression of the retrotransposed transgene may be controlled by a strong promoter that can be regulated in an in vivo environment. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter is a L1-Ta promoter.

In some embodiments, the ORF1p may be overexpressed. In some embodiments, the ORF2 may be overexpressed. In some embodiments, the ORF1p or ORF2p or both are overexpressed. In some embodiments, upon overexpression of an ORF1, ORF1p is at least 1.1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 12 fold, 14 fold, 16 fold, 18 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or at least 100 fold higher than a cell not overexpressing and ORF1.

In some embodiments, upon overexpression of an ORF2 sequence, ORF2p is at least 1.1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 12 fold, 14 fold, 16 fold, 18 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or at least 100 fold higher than a cell not overexpressing and ORF2p.

Retrotransposition Fidelity and Target Specificity

The LINE-1 elements can bind to their own mRNA poly A tail to initiate retrotransposition. LINE-1 elements preferably retrotranspose their own mRNA over random mRNAs (Dewannieux et al., 2013, 3,000-fold higher LINE-1 retrotransposition as compared to random mRNAs). In addition, LINE-1 elements can also integrate non-specific poly-A sequences within a genome.

In one aspect, provided herein are retrotransposition compositions and methods of using the same with increased retrotransposition specificity. For example, retrotransposition compositions with high specificity may be used for highly specific and efficient reverse transcription and subsequently, integration into genome of a target cell, e.g., a myeloid cell. In some embodiments, a retrotransposition composition provided herein comprises a retrotransposition cassette that comprises one or more additional components that increases integration or retrotransposing specificity. For example, the retrotransposon cassette may encode one or more additional elements that allows for high affinity RNA-protein interaction to out compete non-specific binding between poly-A sequences and ORF2.

Accordingly, several measures are disclosed herein for enhancing integration or retrotransposition efficiency.

One exemplary measure for enhancing integration or retrotransposition efficiency is external manipulation of the cells. The endonuclease function of the retrotransposition machinery delivered in a cell may likely be subject to inhibition by the cell's transposition silencing machinery, such as DNA repair pathways. For example, small molecules can be used to modulate or inhibit DNA repair pathways in the cells prior to introducing the nucleic acid. For example, cell sorting and/or synchronization can be used prior to introducing the nucleic acid, such as by electroporation, as cell cycle synchronized cell populations were shown to increase gene transfer to the cells. Cell sorting may be utilized to synchronize or homogenize the cell types and increase uniform transfer and expression of the exogenous nucleic acid. Uniformity may be achieved sorting stem cells from non-stem cells. Another exemplary measure for enhancing integration or retrotransposition efficiency is to enhance biochemical activity. For example, this may be achieved by increasing reverse-transcriptase processivity or DNA cleavage (endonuclease) activity. Another exemplary measure for enhancing integration or retrotransposition efficiency is to subvert endogenous silencing mechanisms. For example, this may be achieved by replacing entire LINE-1 sequence with a different organisms' LINE-1. Another exemplary measure for enhancing integration or retrotransposition efficiency is to enhance translation and ribosome binding. For example, this may be achieved by increasing expression of LINE-1 proteins, increasing LINE protein binding LINE-1 mRNA, or increasing LINE-1 complex binding to ribosomes. Another exemplary measure for enhancing integration or retrotransposition efficiency is to increase nuclear import or retention. For example, this may be achieved by fusing the LINE-1 sequence to a nuclear retention signal sequence. Another exemplary measure for enhancing integration or retrotransposition efficiency is to enhance sequence-specific insertion. For example, this may be achieved by fusing a targeting domain to ORF2 to increase sequence specific retrotransposition.

In one embodiment, the method encompasses enhancing the retrotransposon for increasing specificity and robustness of expression of the cargo by modifying the UTR sequence of the LINE-1 ORFs. In some embodiments, the 5′UTR upstream of ORF1 or ORF2 encoding sequence may be further modified to comprise a sequence that is complementary to the sequence of a target region within the genome that helps in homologous recombination at the specific site where the ORF nuclease can act and the retrotransposition can take place. In some embodiments, the sequence that can bind to a target sequence by homology is between 2-15 nucleotides long. In some embodiments, the sequence having homology to a genomic target that is included in the 5′UTR of an ORF1 mRNA may be about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides or about 10 nucleotides long. In some embodiments, the sequence having homology to a genomic target is about 12 or about 15 nucleotides long. In some embodiments, the sequence having homology to a genomic target is at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 1120 or 125 nucleotides in length. In some embodiments, the sequence having homology to a genomic target comprises about 2-5, about 2-6, about 2-8 or about 2-10, or about 2-12 contiguous nucleotides that share complementarity with the respective target region within the genome. In some embodiments, the sequence having homology to a genomic target is at least about or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 1120 or 125 contiguous nucleotides that share complementarity with the respective target region within the genome.

In some embodiments, an ORF2 is associated with or fused to an additional protein domain that comprises RNA binding activity. In some embodiments, the retrotransposon cassette comprises a cognate RNA sequence that comprises affinity with the additional protein domain associated with or fused to the ORF2. In some embodiments, the ORF2 is associated with or fused to a MS2-MCP coat protein. In some embodiments, the retrotransposon cassette further comprises a MS2 hairpin RNA sequence in the 3′ or 5′ UTR sequence that interacts with the MS2-MCP coat protein. In some embodiments, the ORF2 is associated with or fused to a PP7 coat protein. In some embodiments, the retrotransposon cassette further comprises a PP7 hairpin RNA sequence in the 3′ or 5′ UTR sequence that interacts with the MS2-MCP coat protein. In some embodiments, the one or more additional elements increases retrotransposition specificity by at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 50 fold, at least 100 fold, at least 200 fold, at least 300 fold, at least 500 fold, at least 1000 fold, at least 1500 fold, at least 2000 fold, at least 3000 fold, at least 5000 fold or more as compared to a retrotransposon cassette without the one or more additional elements.

The DNA endonuclease domain appears to have specificity for a series of purines 3′ of the target site followed by a series of pyrimidines (Py)_(n)↓(Pu)_(n). An exemplary sequence may be (Adenosine)_(n)↓(Thymidine)_(n).

In one aspect, provided herein are methods of using retrotransposition having high target specificity. Consequently, provided herein is a method and compositions for stable incorporation of a transgene into the genome of a myeloid cell, such as a monocyte or macrophage, wherein the method comprises incorporating the transgene using a non-LTR retrotransposon system, wherein the retrotransposition occurs at a specific genomic locus with a target specificity, high precision and fidelity. Therefore, in some embodiments, the method comprises administration to the cell a composition comprising a system having at least one transgene, flanked with one or more retrotransposable elements, and one or more nucleic acids encoding one or more proteins for increasing the transposition specificity, and/or further comprising modifying one or more genes associated with the retrotransposition.

The nucleic acid comprising the transgene, situated in 3′ UTR region of the retrotransposable elements is often referred to as a retrotransposition cassette. Accordingly, in some embodiments, the retrotransposition cassette comprises the nucleic acid encoding the transgene and flanking Alu transposable elements. The retrotransposable elements comprise a sequence for binding the retrotransposons, for example, L1-transposons, such as L1-ORF proteins, ORF1p and ORF2p. ORF proteins are known to bind to their own mRNA sequence for retrotransposition. Therefore, the retrotransposition cassette comprises the nucleic acid encoding the transgene; a flanking L1-ORF2p binding sequence, and/or a L1-ORF1p binding sequence, comprising a sequence encoding a L1-ORF1p encoding sequence and a L1-ORF2p encoding sequence outside the transgene sequence. In some embodiments, the L1-ORF1 and L1-ORF2 are interspersed by a spacer region, also termed as an ORF1-ORF2 inter-region. In some embodiments, the L1-ORF1 and L1-ORF2 coding sequences are in an opposite orientation with respect to the coding region of the transgene. The retrotransposition cassette can comprise a poly A region downstream of the L1-ORF2-coding sequence and the transgene sequence is placed downstream of the poly A sequence. The L1-ORF2 comprises a nucleic acid sequence that encodes an endonuclease (EN) and a reverse transcriptase (RT) followed by the poly A sequence. In some embodiments, the L1-ORF2 sequence in the retrotransposition cassette described herein is a complete (intact) sequence, that is, encodes the full length native (WT) L1-ORF2 sequence. In some embodiments, the L1-ORF2 sequence in the retrotransposition cassette described herein comprises a partial or modified sequence.

The system described herein can comprise a promoter for expressing the L1-ORF1p and L1-ORF2p. In some embodiments, the transgene expression is driven by a separate promoter. In some embodiments, the transgene and the ORFs are in tandem orientation. In some embodiments, the transgene and the ORFs are in opposite orientation.

In some embodiments, the method comprises incorporating one or more elements in addition to the retrotransposon cassette. In some embodiments, the one or more additional elements comprise a nucleic acid sequence encoding one or more domains of a heterologous protein. The heterologous protein may be a sequence specific nucleic acid binding protein, for example, a sequence specific DNA binding protein domain (DBD). In some embodiments, the heterologous protein is a nuclease or a fragment thereof. In some embodiments, the additional elements comprise a nucleic acid sequence encoding one or more nuclease domains or fragments thereof from a heterologous protein. In some embodiments, the heterologous nuclease domain has reduced nuclease activity. In some embodiments, the heterologous nuclease domain is rendered inactive. In some embodiments, the ORF2 nuclease is rendered inactive; whereas one or more nuclease domains from the heterologous protein is configured to render specificity to the retrotransposition. In some embodiments, one or more nuclease domains or fragments thereof from the heterologous protein targets a specific desired polynucleotide within the genome where retrotransposition and incorporation of the polynucleotide of interest is to be incorporated. In some embodiments, the one or more nuclease domains from the heterologous protein comprise a mega-TAL nuclease domain, TALENs, or a zinc finger nuclease domain, for example, a mega-TAL, a TALE, or a zinc finger domain fused to or associated with a nuclease domain, e.g., a FokI nuclease domain. In some embodiments, the one or more nuclease domains from the heterologous protein comprise a CRISPR-Cas protein domain loaded with a specific guide nucleic acid, e.g., a guide RNA (gRNA) for a specific target locus. In some embodiments, the CRISPR-Cas protein is a Cas9, a Cas12a, a Cas12b, a Cas13, a CasX, or a CasY protein domain. In some embodiments, the one or more nuclease domains from the heterologous protein has target specificity.

In some embodiments, the additional nuclease domain may be incorporated into the ORF2 domain. In some embodiments, the additional nuclease may be fused with the ORF2p domain. In some embodiments, the additional nuclease domain may be fused to an ORF2p, wherein the ORF2p includes a mutation in the ORF2p endonuclease domain. In some embodiments, the mutation inactivates the ORF2p endonuclease domain. In some embodiments, the mutation is a point mutation. In some embodiments, the mutation is a deletion. In some embodiments, the mutation is an insertion. In some embodiments, the mutation abrogates the ORF2 endonuclease (nickase) activity. In some embodiments, a mutation inactivates the DNA target recognition of ORF2p endonuclease. In some embodiments, the mutation covers a region associated with ORF2p nuclease-DNA recognition. In some embodiments, a mutation reduces the DNA target recognition of ORF2p endonuclease. In some embodiments, the ORF2p endonuclease domain mutation is in the N-terminal region of the protein. In some embodiments, the ORF2p endonuclease domain mutation is in a conserved region of the protein. In some embodiments, the ORF2p endonuclease domain mutation is in the conserved N-terminal region of the protein. In some embodiments, the mutation comprises the N14 amino acid within L1 endonuclease domain. In some embodiments, the mutation comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive amino acids including the N14 amino acid within L1 endonuclease domain. In some embodiments, the mutation comprises the comprises the E43 amino acid within L1 endonuclease. In some embodiments, the mutation comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive amino acids including the E43 amino acid within L1 endonuclease domain. In some embodiments, the mutation comprises 2 or more amino acids in the L1 endonuclease domain including N14, or E43 or a combination thereof. In some embodiments, the mutation comprises D145 of the L1 endonuclease domain. In some embodiments, the mutation may be D145A. In some embodiments, the may be a comprise D205 of the L1 endonuclease domain. In some embodiments, the mutation may be D205G. In some embodiments, the mutation may comprise H230 of L1 endonuclease domain. In some embodiments, the may be a comprise S228 of the L1 endonuclease domain. In some embodiments, the mutation may be S228P.

In some embodiments, a mutation reduces the DNA target recognition of ORF2p endonuclease by at least 50%. In some embodiments, a mutation reduces the DNA target recognition of ORF2p endonuclease by at least 60%. In some embodiments, a mutation reduces the DNA target recognition of ORF2p endonuclease by at least 70%. In some embodiments, a mutation reduces the DNA target recognition of ORF2p endonuclease 80%. In some embodiments, a mutation reduces the DNA target recognition of ORF2p endonuclease 90%. In some embodiments, a mutation reduces the DNA target recognition of ORF2p by 95%. In some embodiments, a mutation reduces the DNA target recognition of ORF2p by 100%.

In some embodiments, the mutation is a deletion. In some embodiments, the deletion is complete, i.e., 100% of the L1 endonuclease domain is deleted. In some embodiments, the deletion is partial. In some embodiments, the about 98%, about 95%, about 94%, about 93%, about 92% about 91%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, or about 50% of the ORF2 endonuclease domain is deleted.

In some embodiments, an additional nuclease domain is inserted into the ORF2 protein sequence. In some embodiments, ORF2 endonuclease domain is deleted, and is replaced with an endonuclease domain from a heterologous protein. In some embodiments, the ORF2 endonuclease is partially deleted and replaced with an endonuclease domain from a heterologous protein. The endonuclease domain from a heterologous protein may be a mega-TAL nuclease domain. The endonuclease domain from a heterologous protein may be a TALENs. The endonuclease domain from a heterologous protein may be a Cas9 loaded with a specific gRNA for a locus.

In some embodiments, the endonuclease is an endonuclease that has (i) a specific target on the genome and (ii) it creates a 5′-P and a 3′-OH terminus at the cleavage site.

In some embodiments, the additional endonuclease domain from a heterologous protein is an endonuclease domain from a related retrotransposon.

In some embodiments, the endonuclease domain from a heterologous protein may comprise a bacterial endonuclease engineered for targeting a specific site. In some embodiments, the endonuclease domain from a heterologous protein may comprise a domain of a homing endonuclease or a fragment thereof. In some embodiments, the endonuclease is a homing endonuclease. In some embodiments, the homing endonuclease is an engineered LAGLIDADG (SEQ ID NO: 62) homing endonucleases (LHEs) or a fragment thereof. In some embodiments, additional endonucleases may be a restriction endonuclease, Cre, Cas TAL or fragments thereof. In some embodiments, the endonuclease may comprise a Group II intron encoded protein (ribozyme) or a fragment thereof.

An engineered or modified L1-ORF2p as discussed in the preceding paragraphs, that is endowed with specific DNA targeting capability due to the additional/heterologous endonuclease is expected to be highly advantageous in driving targeted stable integration of a transgene into the genome. The engineered L1-ORF2p can generate much reduced off-target effects when expressed in a cell than using a native, non-engineered L1-ORF2p. In some embodiments, the engineered L1-ORF2p generates no off-target effect.

In some embodiments, the engineered or modified L1-ORF2p targets a recognition site that is other than the usual (Py)_(n)↓(Pu)_(n) site. In some embodiments, engineered L1-ORF2p targets a recognition site that comprises the (Py)_(n)↓(Pu)_(n) site, for example, TTTT/AA site, such as a hybrid target site. In some embodiments, the engineered L1-ORF2p targets a recognition site having at least one nucleotide in addition to the conventional L1-ORF2 (Py)_(n)↓(Pu)_(n) site, for example TTTT/AAG, or TTTT/AAC, or TTTT/AAT, TTTT/AAA, GTTTT/AA, CTTTT/AA, ATTTT/AA, or TTTTT/AA. In some embodiments, the engineered L1-ORF2p targets a recognition site that is in addition to the conventional L1-ORF2p (Py)_(n)↓(Pu)_(n) site. In some embodiments, the engineered L1-ORF2p targets a recognition site that is other than to the conventional L1-ORF2p (Py)_(n)↓(Pu)_(n) site. In some embodiments, the engineered L1-ORF2p targets a recognition site that is 4, 5, 6, 7, 8, 9, 10 or more nucleotides long. In some embodiments, the engineered or modified L1-ORF2p recognition site may be 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.

The engineered L1-ORF2p can be engineered to retain its ability to bind to its own mRNA after translation and reverse transcribe with high efficiency. In some embodiments, the engineered L1-ORF2p has enhanced efficiency of reverse transcription compared to a native (WT) L1-ORF2p.

In some embodiments, the system comprising a retrotransposable element further comprises a gene modification that reduces non-specific retrotransposition. In some embodiments, the gene modification may comprise a sequence encoding the L1-ORF2p. In some embodiments, the modification may comprise mutation of one or more amino acids that are essential for binding to a protein that helps ORF2p binding to the target genomic DNA. A protein that helps ORF2p binding to the target genomic DNA may be part of the chromatin-ORF interactome. In some embodiments, the modification may comprise one or more amino acids that are essential for binding to a protein that helps ORF2p DNA endonuclease activity. In some embodiments, the modification may comprise one or more amino acids that are essential for binding to a protein that helps ORF2p RT activity. In some embodiments, the modification may comprise at a protein binding site on ORF2p such that the association of a protein with ORF2p is altered, wherein binding of the protein to ORF2p is required for binding to chromatin. In some embodiments, the modification may comprise at a protein binding site on ORF2p such that the association of the protein with ORF2p is more stringent and/or specific than in absence of the modification. In some embodiments, as a consequence of altered association of ORF2p with the protein owing to the modification of ORF2p coding sequence at the protein binding site, the binding of ORF2p to the target DNA has increased specificity. In some embodiments, the modification may reduce binding of ORF2 to one or more proteins that are part of the ORF2p chromatin interactome.

In some embodiments, the gene modification may be in the PIP domain of ORF2p.

In some embodiments, the gene modification may be in one or more genes encoding a protein that binds to an ORF2p and helps in the recognition, binding, endonuclease or RT activity of ORF2p. In some embodiments, the gene modification may be in one or more genes encoding PCNA, PARP1, PABP, MCM, TOP1, RPA, PURA, PURB, RUVBL2, NAP1, ZCCHC3, UPF1 or MOV10 proteins at an ORF2p interacting site for each protein or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the modification may be on an ORF2p binding domain of PCNA at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the modification may be on an ORF2p binding domain of TOP1. In some embodiments, the modification may be on an ORF2p binding domain of RPA. In some embodiments, the modification may be on an ORF2p binding domain of PARP1 at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the modification may be on an ORF2p binding domain of PABP (e.g., PABPC1) at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the gene modification may be on an MCM gene. In some embodiments, the gene modification may be on a gene encoding MCM3 protein at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the gene modification may be on a gene encoding MCM5 protein at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the gene modification may be on a gene encoding MCM6 protein at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the gene modification may be on a gene encoding MEPCE protein at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the gene modification may be on a gene encoding on a gene encoding RUVBL1 or RUVBL2 protein at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA. In some embodiments, the gene modification may be on a gene encoding on a gene encoding TROVE protein at an ORF2p interacting site or at a site that affects the protein's interaction with ORF2p or the interaction of ORF2p with target DNA.

In some embodiments, the retrotransposition system disclosed herein comprises one or more elements that increase the fidelity of reverse transcription.

In some embodiments, the L1-ORF2 RT domain is modified. In some embodiments, the modification includes one or more of: increasing fidelity, increasing processivity, increasing DNA-RNA substrate affinity; or inactivating RNase H activity.

In some embodiments, the modification comprises introducing one or more mutations in the RT domain of the L1-ORF2, such that the fidelity of the RT is increased. In some embodiments, the mutation comprises a point mutation. In some embodiments, the mutation comprises alteration, such as substitution of one, two three, four, five, six or more amino acids in the L1-ORF2p RT domain. In some embodiments, the mutation comprises deletion of one or more amino acids, for example, one, two, three, four, five, six, seven, eight, nine, ten or more amino acids in the L1-ORF2p RT domain. In some embodiments, the mutation may comprise an in-del mutation. In some embodiments, the mutation may comprise a frame-shift mutation.

In some embodiments, the modification may comprise inclusion of an additional RT domain or fragment thereof from a second protein. In some embodiments, the second protein is a viral reverse transcriptase. In some embodiments, the second protein is a non-viral reverse transcriptase. In some embodiments, the second protein is a retrotransposable element. In some embodiments, the second protein is a non-LTR retrotransposable element. In some embodiments, the second protein is a group II intron protein. In some embodiments, the group II intron is as TGIRTII. In some embodiments, the second protein is a Cas nickase, wherein the retrotransposable system further comprises introducing a guide RNA. In some embodiments, the second protein is a Cas9 endonuclease, wherein the retrotransposable system further comprises introducing a guide RNA. In some embodiments, the second protein or fragment thereof is fused to the N-terminus of the L1-ORF2 RT domain or the modified L1-ORF2 RT domain. In some embodiments, the second protein or fragment thereof is fused to the C-terminus of the L1-ORF2 RT domain or the modified L1-ORF2 RT domain.

In some embodiments, the additional RT domain or fragment thereof from the second protein is incorporated in the retrotransposition system in addition to the full-length WT L1-ORF2p RT domain. In some embodiments, the additional RT domain or fragment thereof from the second protein is incorporated in presence of a modified (engineered) L1-ORF2p RT domain or a fragment thereof, where the modification (or engineering) may comprise a mutation for enhancement of the L1-ORF2p RT processivity, stability and/or fidelity of the modified L1-ORF2p RT compared to the native or WT ORF2p.

In some embodiments, the reverse transcriptase domain could be replaced with other more highly processive and high-fidelity RT domains from other retroelements or group II introns, such as TGIRTII.

In some embodiments, the modification may comprise a fusion with an additional RT domain or fragment thereof from a second protein. In some embodiments, the second protein may comprise a retroelement. The additional RT domain or fragment thereof from a second protein is configured to increase the fidelity of reverse transcription of the fused L1-ORF2p RT domain. In some embodiments, the nucleic acid encoding the additional RT domain or fragment thereof is fused to a native or WT L1-ORF2 encoding sequence. In some embodiments, the nucleic acid encoding the additional RT domain or fragment thereof from a second protein is fused to a modified L1-ORF2 encoding sequence. In some embodiments, the modification comprises introducing one or more mutations in the RT domain of the L1-ORF2 or fragment thereof, such that the fidelity of the fused RT is increased. In some embodiments, the mutation in the RT domain of the L1-ORF2 or fragment thereof comprises a point mutation. In some embodiments, the mutation comprises alteration, such as substitution of one, two three, four, five, six or more amino acids in the L1-ORF2p RT domain. In some embodiments, the mutation comprises deletion of one or more amino acids, for example, one, two, three, four, five, six, seven, eight, nine, ten or more amino acids in the L1-ORF2p RT domain. In some embodiments, the mutation may comprise an in-del mutation. In some embodiments, the mutation may comprise a frame-shift mutation.

In some embodiments, the modified L1-ORF2p RT domain has increased processivity than the WT L1-ORF2p RT domain.

In some embodiments, the modified L1-ORF2p RT domain has at least 10% higher processivity and/or fidelity over the WT L1-ORF2p RT domain. In some embodiments, the modified L1-ORF2p RT domain has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 1000% or higher processivity and/or fidelity over the WT L1-ORF2p RT domain. In some embodiments, the modified RT can process greater than 6 kb nucleic acid stretch. In some embodiments, the modified RT can process greater than 7 kb nucleic acid stretch. In some embodiments, the modified RT can process greater than 8 kb nucleic acid stretch. In some embodiments, the modified RT can process greater than 9 kb nucleic acid stretch. In some embodiments, the modified RT can process greater than 10 kb nucleic acid stretch.

B. Group II Introns and Ribozymes

Group II enzymes are mobile ribozymes that self-splice precursor RNAs, yielding excised intron lariat RNAs. The introns encode a reverse transcriptase. The reverse transcriptase may stabilize the RNA for forward and reverse splicing, and later in converting the integrated intron RNA to DNA.

Group II RNAs are characterized by a conserved secondary structure spanning 400-800 nucleotides. The secondary structure is formed by six domains DI-VI, and is organized in a structure resembling a wheel, where the domains radiate from a central point. The domains interact to form a conserved tertiary structure that brings together distant sequences to form an active site. The active site binds the splice sites and branch point residue nucleotide and in association of Mg2+ cations, activate catalysis of splicing. The DV domain is within the active site, which has the conserved catalytic AGC and an AY bulge and both these regions bind Mg2+ ions necessary for the catalysis. DI is the largest domain with upper and lower halves separated by kappa and zeta motifs. The lower half contains the ε′ motif, which is associated with an active site. The upper half contains sequence elements that bind to the 5′ and 3′ exons at the active sites. DIV encodes the intron-encoded protein (IEP) with subdomain IVa near the 5′-end containing the high affinity binding site for IEP. Group II introns have conserved 5′- and 3′-end sequences, GUGYG and AY respectively.

Group II RNA introns can be utilized to retrotranspose a sequence of interest into DNA via target primed reverse transcription. This process of transposition by Group II RNA introns is often referred to as retrohoming. Group II introns recognize DNA target sites by base pairing of the intron RNA to the DNA target sequence, they can be modified to retarget a specific sequence carried within the intron to a desired DNA site.

In some embodiments, the method and compositions for retrotransposition described herein may comprise a Group II intron sequence, a modified Group II intron sequence or a fragment thereof. Exemplary Group II IEPs (maturase) include but are not limited to bacterial, fungal, yeast IEPs, that are functional in human cells. In particular, the nuclease leaves a 3′-OH at the cleavage site of the DNA which can be utilized by another RT for priming and reverse transcription. An exemplary Group II maturase may be TGIRT (thermally stable group II intron maturase).

In one or more embodiments of several aspects described herein, the nucleic acid construct comprises an RNA. In one or more embodiments of several aspects of the disclosure, the nucleic acid construct is an RNA. In one or more embodiments of several aspects of the disclosure, the nucleic acid construct is an mRNA. In one aspect, the mRNA comprises a sequence of a heterologous gene or portion thereof, wherein the heterologous gene or portion thereof encodes a polypeptide or protein. In some embodiments, the mRNA comprises a sequence encoding a fusion protein. In some embodiments, the mRNA comprises a sequence encoding a recombinant protein. In some embodiments, the mRNA comprises a sequence encoding a synthetic protein. In some embodiments, the nucleic acid comprises one or more sequences, wherein the one or more sequences encode on or more heterologous proteins, one or more recombinant proteins, or one or more synthetic proteins or a combination thereof. In some embodiments, the nucleic acid comprises one or more sequences, wherein the one or more sequences encode on or more heterologous proteins comprising a synthetic protein or a recombinant protein. In some embodiments, the synthetic or recombinant protein is a recombinant fusion protein.

In one or more of embodiments of several aspects of the disclosure, the nucleic acid construct is developed for expressing in a eukaryotic cell. In some embodiments, the nucleic acid construct is developed for expressing in a human cell. In some embodiments, the nucleic acid construct is developed for expressing in a hematopoietic cell. In some embodiments, the nucleic acid construct is developed for expressing in a myeloid cell. In some embodiments, the myeloid cell is a human cell.

II. Modifications in Nucleic Acid Constructs for Methods of Enhancement of Expression of Encoded Protein

In some aspects of the disclosure, the recombinant nucleic acid is modified for enhanced expression of the protein encoded by a sequence of the nucleic acid. Enhanced expression of the protein encoded therein can be a function of the nucleic acid stability, translation efficiency and the stability of the translated protein. A number of modifications are contemplated herein for incorporation in the design of the nucleic acid construct that can confer nucleic acid stability, such as stability of the messenger RNA encoding the exogenous or heterologous protein, which may be a synthetic recombinant protein or a fragment thereof.

In some embodiments, the nucleic acid is mRNA, comprising one or more sequences, wherein the one or more sequences encode one or more heterologous proteins comprising a synthetic or a recombinant fusion protein.

In some embodiments, one or more modifications are made in the mRNA comprising a sequence encoding a recombinant or fusion protein to increase the mRNA half-life.

Structural Elements to Block 5′- and 3′-Degradations by Exonucleases: 5′-Cap and 3′ UTR Modifications

A proper 5′-cap structure is important in the synthesis of functional messenger RNA. In some embodiments, the 5′-cap comprises a guanosine triphosphate arranged as GpppG at the 5′ terminus of the nucleic acid. In some embodiments, the mRNA comprises a 5′ 7-methylguanosine cap, m7-GpppG. A 5′ 7-methylguanosine cap increases mRNA translational efficiency and prevents degradation of mRNA 5′-3′ exonucleases. In some embodiments, the mRNA comprises “anti-reverse” cap analog (ARCA, m^(7,3′-O)GpppG). Translational efficiency, however, can be markedly increased by usage of the ARCA. In some embodiments, the guanosine cap is a Cap 0 structure. In some embodiments, the guanosine cap is a Cap 1 structure. In addition to its essential role of cap-dependent initiation of protein synthesis, the mRNA cap also functions as a protective group from 5′ to 3′ exonuclease cleavage and a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation and nuclear export. It acts as the anchor for the recruitment of initiation factors that initiate protein synthesis and the 5′ to 3′ looping of mRNA during translation. Three enzymatic activities are required to generate the Cap 0 structure, namely, RNA triphosphatase (TPase), RNA guanylyltransferase (GTase) and guanine-N7 methyltransferase (guanine-N7 MTase). Each of these enzyme activities carries out an essential step in the conversion of the 5′ triphosphate of nascent RNA to the Cap 0 structure. RNA TPase removes the γ-phosphate from the 5′ triphosphate to generate 5′ diphosphate RNA. GTase transfers a GMP group from GTP to the 5′ diphosphate via a lysine-GMP covalent intermediate. The guanine-N7 MTase then adds a methyl group to the N7 amine of the guanine cap to form the cap 0 structure. For Cap 1 structure, m7G-specific 2′O methyltransferase (2′O MTase) methylates the +1 ribonucleotide at the 2′O position of the ribose to generate the cap 1 structure. The nuclear RNA capping enzyme interacts with the polymerase subunit of RNA polymerase II complex at phosphorylated Ser5 of the C-terminal heptad repeats. RNA guanine-N7 methyltransferase also interacts with the RNA polymerase II phosphorylated heptad repeats. In some embodiments, the cap is a G-quadruplex cap.

In some embodiments, the mRNA is synthesized by in vitro transcription (IVT). In some embodiments, mRNA synthesis and capping may be performed in one step. Capping may occur in the same reaction mixture as IVT. In some embodiments, mRNA synthesis and capping may be performed in separate steps. mRNA thus formed by IVT is purified and then capped.

In some embodiments, the nucleic acid construct, e.g., the mRNA construct, comprises one or more sequences encoding a protein or a polypeptide of interest can be designed to comprise elements that protect, prevent, inhibit or reduce degradation of the mRNA by endogenous 5′-3′ exoribonucleases, for example, Xrn1. Xrn1 is a cellular enzyme in the normal RNA decay pathways that degrades 5′ monophosphorylated RNAs. However, some viral RNA structural elements are found to be particularly resistant to such RNases, for example, the Xrn1-resistant structure in flaviviral sfRNAs, called the ‘xrRNA’. For example, the mosquito-borne flaviviruses (MBFV) genomes contain discrete RNA structures in their 3′-untranslated region (UTR) that block the progression of Xrn1. These RNA elements are sufficient to block Xrn1 without the use of accessory proteins. xrRNAs halt the enzyme at a defined location such that the viral RNA located downstream of the xrRNAs is protected from degradation. The xrRNAs from Zika virus or Murray Valley encephalitis virus, for example, comprise three-way junction and multiple pseudoknot interactions that create an unusual and complex fold that requires a set of nucleotides conserved across the MBFVs structure. xrRNAs halt the enzyme at a defined location such that the viral RNA located downstream of the xrRNAs is protected from degradation. The 5′-end of the RNA passes through a ring-like structure of the fold and is believed to remain protected from the Xrn1-like exonuclease.

In some embodiments, the nucleic acid construct comprising the one or more sequences that encode a protein of interest may comprise one or more xrRNA structures incorporated therein. In some embodiments, the xrRNA is a stretch of nucleotides having the conserved regions of the 3′ UTR of one or more viral xrRNA sequences. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more xrRNA elements are incorporated within the nucleic acid construct. In some embodiments, 2 or more xrRNA elements are incorporated in tandem within the nucleic acid construct. In some embodiments, the xrRNA comprise one or more regions comprising conserved sequences or fragments thereof or modifications thereof. In some embodiments, the xrRNA is placed at the 3′UTR of a retrotransposon element. In some embodiments, the xrRNA is placed at upstream of the sequences encoding the one or more proteins or polypeptides. In some embodiments, the xrRNA is placed in the 3′UTR of a retrotransposon element, such as an ORF2 sequence, and upstream of the sequences encoding the one or more proteins or polypeptides.

In some embodiments, the xrRNA structure comprises a MBFV xrRNA sequence, or a sequence that is at least 90% identical thereof. In some embodiments, the xrRNA structure comprises a tick-borne flaviviruses (TBFVs) xrRNA sequence, or a sequence that is at least 90% identical thereof. In some embodiments, the xrRNA structure comprises a tick-borne flaviviruses (TBFVs) xrRNA sequence, or a sequence that is at least 90% identical thereof. In some embodiments, the xrRNA structure comprises a tick-borne flaviviruses (TBFVs) xrRNA sequence, or a sequence that is at least 90% identical thereof. In some embodiments, the xrRNA structure comprises a xrRNA sequence from a member of no known arthropod vector flaviviruses (NKVFVs), or a sequence that is at least 90% identical thereof. In some embodiments, the xrRNA structure comprises a xrRNA sequence from a member of insect-specific flaviviruses (ISFVs), or a sequence that is at least 90% identical thereof. In some embodiments, the xrRNA structure comprises a Zikavirus xrRNA sequence, or a sequence that is at least 90% identical thereof. It is hereby contemplated that any known xrRNA structural elements or conceivable non-obvious variations thereof may be used for the purpose described herein.

Several messenger RNAs from different organisms exhibit one or more pseudoknot structures that exhibits resistance from 5′-3′ exonuclease. A pseudoknot is a RNA structure that is minimally composed of two helical segments connected by single-stranded regions or loops. Although several distinct folding topologies of pseudoknots exist.

Poly A Tail Modifications

The poly A structure in the 3′UTR of an mRNA is an important regulator of mRNA half-life. Deadenylation of the 3′ end of the poly A tail is the first step of the intracellular mRNA degradation. In some embodiments, the length of the poly A tail of the mRNA construct is taken into critical consideration and designed for maximizing the expression of the protein encoded by the mRNA coding region, and the mRNA stability. In some embodiments, the nucleic acid construct comprises one or more poly A sequences. In some embodiments, the poly A sequence at the 3′UTR of the sequences encoding the one or more proteins or polypeptides comprise 20-200 adenosine nucleobases. In some embodiments, the poly A sequence comprises 30-200 adenosine nucleobases. In some embodiments, the poly A sequence comprises 50-200 adenosine nucleobases. In some embodiments, the poly A sequence comprises 80-200 adenosine nucleobases. In some embodiments, the mRNA segment comprising the sequences that encode one or more proteins or polypeptides comprises a 3′-UTR having a poly-A tail comprising about 180 adenosine nucleobases, or about 140 adenosine nucleobases, or about 120 adenosine nucleobases. In some embodiments, the poly A tail comprises about 122 adenosine nucleobases. In some embodiments, the poly A sequence comprises 50 adenosine nucleobases. In some embodiments, the poly A sequence comprises 30 adenosine nucleobases. In some embodiments, the adenosine nucleobases in the poly A tail are placed in tandem, with or without intervening non-adenosine bases. In some embodiments, one or more non-adenosine nucleobases are incorporated in the poly A tail, which confer further resistance to certain exonucleases.

In some embodiments, the stretch of adenosines in poly A tail of the construct comprises one or more non-adenosine (A) nucleobase. In some embodiments, the non-A nucleobase is present at −3, −2, −1, and/or +1 position at the poly A 3′-terminal region. In some embodiments, the non-A bases comprise a guanosine (G) or a cytosine (C) or an uracil base (U). In some embodiments, the non-A base is a G. In some embodiments, the non-A base more than one, in tandem, for example, GG. In some embodiments, the modification at the 3′ end of the poly A tail with one or more non-A base is directed at disrupting the A base stacking at the poly A tail. The poly A base stacking promotes deadenylation by various deadenylating enzymes, and therefore 3′ end of poly A tail ending in -AAAG, -AAAGA, or -AAAGGA are effective in conferring stability against deadenylation. In some organisms, a GC sequence intervening a poly A sequence is shown to effectively show down 3′-5′ exonuclease mediated decay. A modification contemplated herein comprises an intervening non-A residue, or a non-A residue duplex intervening a poly A stretch at the 3′ end.

In some embodiments, a triplex structure is introduced in the 3′ UTR which effectively stalls or slows down exonuclease activity involving the 3′ end.

In some embodiments, the mRNA with the modifications described above has an extended half-life and demonstrates stable expression over a longer period than the unmodified mRNA. In some embodiments, the mRNA stably expresses for greater than 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days 9 days or 10 days or more, and the mRNA or its protein product is detectable in vivo. In some embodiments, the mRNA is detected up to 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or 15 days in vivo. In some embodiments, a protein product of the mRNA is detected up to 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 20 days, 25 days or 30 days in vivo.

CircRNA and tectoRNA

Circular RNA is useful in the design and production of stable form of RNA used as a messenger RNA to direct synthesis protein chains, such as long, multiply repeating protein chains There are few methods to make circular RNA (circRNA). They include protein-mediated ligation of RNA ends using RNA ligase and using a split self-splicing intron, such that if the two halves of the intron are located and the ends of a transcribed mRNA, the intron will splice itself out and leave a ligated product (FIG. 3A). Another technique relies on the ability of T4 DNA ligase to act as an RNA ligase when the RNA ends to be ligated are held together by an oligonucleotide. Both these techniques suffer from inefficiency and require a large amount of enzyme. A third technique uses the cyclization or circularization activity of group I introns where most of the intron sequences that carry out the reaction must remain a part of the circle. Group I introns share a complex set of secondary and tertiary structures containing a series of conserved RNA stem loops which form the catalytic core. Many of these introns are self-splicing in vitro and can splice and form two ligated exons as RNA with no accessory protein factors. The products created by the group I autocatalytic reaction are (1) an upstream exon ligated at the 5′ splice site to the 3′ splice site of a downstream exon and (2) a linear intron that can undergo further reversible auto-catalysis to form a circular intron. The presence of such a large highly structured nucleic acid sequence severely limits the types of RNA sequences that can be made circular by that technique. In addition, the catalytic activity of the intron may remain and interfere with structure and function of the circular RNA.

It is useful to increase the rate of the reaction, and thus the overall efficiency, by bringing the ends of the RNA in closer proximity. Previous work has achieved this by including complementary RNA sequences 3′ and 5′ to the ends of the mRNA such that upon hybridization of these sequences, the ends of the mRNA are in closer proximity such that it can undergo the ligation or self-splicing reaction with an overall faster rate compared to without the complementary sequences. These are called homology arms (FIG. 3A) of the self-splicing version of the circularization reaction. A major issue with such hybridization strategy is that if there are complementary sequences within the coding region to either of the homology arms, hybridization would actually inhibit the splicing reaction and the arms would need to be optimized for each new coding region. An alternative to this strategy described herein is the use of RNA sequences that fold a three-dimensional structure to form a stable binding interaction that is independent of sequence.

Non-Watson-Crick RNA tertiary interactions can be exploited to construct ‘tectoRNA’ molecular units, defined as RNA molecules capable of self-assembly. The use of such type of tertiary interactions allows one to control and modulate the assembly process by manipulating cation concentration (e.g. Mg²⁺), and/or suitable temperature and employing modularly designed ‘selector’ RNA molecules. For the self-assembly of one-dimensional arrays, a basic modular unit was designed that comprises a 4-way junction with an interacting module on each helical arm. In some embodiments, the interacting module is a GAAA loop or a specific GAAA loop receptor. Each tectoRNA can interact with two other tectoRNAs via the formation of four loop-receptor interactions, two with each partner molecule.

In some embodiments, the tectoRNA structures are suitably selected, and integrated in the RNA comprising the exon and intron to form a circRNA. In some embodiments, the integration is done by well-known molecular biology techniques such as ligation. In some embodiments, the tectoRNA forms a stable structure at high temperatures. The tectoRNA structure do not compete with internal RNA sequences, thereby creating high efficiency circularization and splicing.

The circRNA can comprise a coding sequence described in any of the preceding sections. For example, it can comprise a sequence encoding fusion protein comprising a tethering or a receptor molecule. The receptor can be a phagocytic receptor fusion protein.

In some embodiments, the intron is a self-splicing intron.

In some embodiments, the terminal regions having the tertiary structures, also termed scaffolding regions for the circRNA, are about 30 nucleotides to about 100 nucleotides long. In some embodiments, the tertiary structure motif is about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, about 60 nucleotides, about 65 nucleotides, about 70 nucleotides or about 75 nucleotides long. In some embodiments, the tertiary motifs are formed at high temperatures. In some embodiments, the tertiary motifs are stable.

In some embodiments, the nucleic acid construct having the one or more modifications as described herein and comprising one or more sequences encoding one or more proteins or polypeptides, is stable when administered in vivo. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA comprising one or more sequences encoding one or more proteins or polypeptides is stable in vivo for more than 2 days, for more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, more than 10 days, more than 11 days, more than 12 days, more than 13 days, more than 14 days, more than 15 days, more than 16 days, more than 17 days, more than 18 days, more than 19 days, or more than 20 days. In some embodiments, the protein encoded by the sequences in the mRNA can be detected in vivo at greater than 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the protein encoded by the sequences in the mRNA can be detected in vivo for about 7 days after the mRNA is administered. In some embodiments, the protein encoded by the sequences in the mRNA can be detected in vivo for about 14 days after the mRNA is administered. In some embodiments, the protein encoded by the sequences in the mRNA can be detected in vivo for about 21 days after the mRNA is administered. In some embodiments, the protein encoded by the sequences in the mRNA can be detected in vivo for about 30 days after the mRNA is administered. In some embodiments, the protein encoded by the sequences in the mRNA can be detected in vivo for more than about 30 days after the mRNA is administered.

In some aspects, enhancing nucleic acid uptake or incorporation within the cell is contemplated for enhancing expression of the retrotransposition. One of the methods include obtaining a homogenous population of cells to initiate incorporation of the nucleic acid, e.g. via transfection, in case of plasmid vector constructs, or via electroporation or any other means that may be used suitably to deliver a nucleic acid molecule into the cell. In some embodiments, cell cycle synchronization may be sought. Cell cycle synchronization may be accomplished by sorting cells for a certain common phenotype. In some embodiments, the cell population may be subjected to a treatment with a reagent that can stall cell cycle progression of all cells at a certain stage. Exemplary reagents can be found in commercial databases, such as www.tocris.com/cell-biology/cell-cycle-inhibitors, or www.scbt.com/browse/chemicals-Other-Chemicals-cell-cycle-arresting-compounds. For example, itraconazole or nocodazole inhibits cell cycle at G1 phase, or reagents that arrest cell cycle at G0/G1 phase, for example, 5-[(4-Ethylphenyl)methylene]-2-thioxo-4-thiazolidinone (compound 10058-F4) (Tocris Bioscience); or a G2M cell cycle blocker, such as AZD 5438 (chemical name, 4-[2-Methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine) which blocks cell cycle at G2M, G1 or S phases, to name a few. Cyclosporin, hydroxyurea, thymidine, are well known reagents that can cause cell cycle arrests. Some reagents may irreversibly alter a cell state or may be toxic for the cells. Serum deprivation of cells for about 2-16 hours prior to electroporation or transfection, depending on the cell type, may also be an easy and reversible strategy for cell synchronization.

In some embodiments, retrotransposition efficiency may be increased by encouraging generation of DNA double stranded breaks to a cell that has been transfected with or electroporated with the retrotransposition constructs as described herein and/or modulating the DNA repair machinery. Application of these techniques may be limited depending on end uses of the cell that would undergo the genetic manipulation ex vivo for stable incorporation of a nucleic acid sequence by this method. In some cases, use of such techniques may be contemplated where robust expression of the protein or transcript encoded by the incorporated nucleic acid is expected as an outcome for a determined period of time. Method of introducing double stranded breaks in a cell include subjecting the cell to controlled ionizing radiation of about 0.1 Gy or less for a short period.

In some embodiments, efficiency of LINE-1 mediated retrotransposition may be increased by treating the cell with small molecule inhibitors of DNA repair proteins to increase the window for the reverse transcriptase to act. Exemplary small molecule inhibitors of DNA repair proteins may be Benzamide (CAS 55-21-0), Olaparib (Lynparza) (CAS 763113-22-0), Rucaparib (Clovis-AG014699, PF-01367338 Pfizer), Niraparib (MK-827 Tesaro) CAS 1038915-60-4); Veliparib (ABT-888 Abbvie) (CAS 912444-00-9); Camptothecin (CPT) (CAS 7689-03-4); Irinotecan (CAS 100286-90-6); Topotecan (Hycamtin® GlaxoSmithKline) (CAS 123948-87-8); NSC 19630 (CAS 72835-26-8); NSC 617145 (CAS 203115-63-3); ML216 (CAS 1430213-30-1); 6-hydroxyDL-dopa (CAS 21373-30-8); D-103; D-G23; DIDS (CAS 67483-13-0); B02 (CAS 1290541-46-6); RI-1 (CAS 415713-60-9); RI-2 (CAS 1417162-36-7); Streptonigrin (SN) (CAS 3930-19-6).

III. Nucleic Acid Cargo: A. Transgene

In one aspect the transgene or noncoding sequence that is the heterologous nucleic acid sequence to be inserted within the genome of a cell is delivered as an mRNA. The mRNA may comprise greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 bases. In some embodiments, the mRNA may be more than 10,000 bases long. In some embodiments, the mRNA may be about 11,000 bases long. In some embodiments, the mRNA may be about 12,000 bases long. In some embodiments, the mRNA comprises a transgene sequence that encodes a fusion protein. In some embodiments, the nucleic acid is delivered as a plasmid.

In some embodiments, the nucleic acid is delivered in the cell by transfection. In some embodiments, the nucleic acid is delivered in the cell by electroporation. In some embodiments, the transfection or electroporation is repeated more than once to enhance incorporation of the nucleic acid into the cell.

Contemplated herein are retrotransposon mediated stable integration of a recombinant nucleic acid encoding a phagocytic or tethering receptor (PR) fusion protein (CFP). In some embodiments, the CFPs comprise: a PR subunit comprising: a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain; and an extracellular domain comprising an antigen binding domain specific to an antigen of a target cell; wherein the transmembrane domain and the extracellular domain are operatively linked.

In some embodiments, the nucleic acid comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising an extracellular domain comprising a CD5 binding domain, and a transmembrane domain operatively linked to the extracellular domain. In some embodiments, the CD5 binding domain is a CD5 binding protein, such as an antigen binding fragment of an antibody, a Fab fragment, an scFv domain or an sdAb domain. In some embodiments, wherein the CD5 binding domain comprises an scFv comprising (i) a variable heavy chain (VH) sequence with at least 90% sequence identity to EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTV (SEQ ID NO: 63); and (ii) a variable light chain (VL) sequence with at least 90% sequence identity to DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSG SGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 64). In some embodiments, the CFP further comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein a wild-type protein comprising the intracellular domain does not comprise the extracellular domain. In some embodiments, the one or more intracellular signaling domains comprises a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, FcαR, and Bai1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcγR, FcαR or FcεR. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain with at least 90% sequence identity to LYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPP (SEQ ID NO: 65). In some embodiments, the one or more intracellular signaling domains further comprises a proinflammatory signaling domain. In some embodiments, the proinflammatory signaling domain comprises a PI3-kinase (PI3K) recruitment domain. In some embodiments, the proinflammatory signaling domain comprises a sequence with at least 90% sequence identity to YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM (SEQ ID NO: 66). In some embodiments, the proinflammatory signaling domain is derived from an intracellular signaling domain of CD40. In some embodiments, the proinflammatory signaling domain comprises a sequence with at least 90% sequence identity to KVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ ID NO: 67). In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a sequence with at least 90% sequence identity to IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 68). In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operatively linked to the transmembrane domain and the CD5 binding domain. In some embodiments, the extracellular domain comprises a sequence with at least 90% sequence identity to ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD (SEQ ID NO: 69). In some embodiments, the CFP comprises an extracellular domain comprising a scFv that specifically binds CD5, and a hinge domain derived from CD8; a hinge domain derived from CD28 or at least a portion of an extracellular domain from CD68; a CD8 transmembrane domain, a CD28 transmembrane domain or a CD68 transmembrane domain; and an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: a first intracellular signaling domain derived from FcγR or FcεR, and a second intracellular signaling domain comprising a PI3K recruitment domain, or derived from CD40. In some embodiments, the recombinant polynucleic acid is an mRNA or circRNA. In some embodiments, the nucleic acid is delivered into a myeloid cell. In some embodiments, the nucleic acid is delivered into a CD14+ cell, a CD14+CD16− cell, an M0 macrophage, an M2 macrophage, an M1 macrophage or a mosaic myeloid cell/macrophage. In some embodiments, the fusion protein comprises a sequence with at least 90% sequence identity to EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGGGGSGG GGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESG VPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKSGGGGSGALSNSIMYF SHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDIYIWAPLAGTCGV LLLSLVITLYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPPQGSGSYEDMRGI LYAAPQLRSIRGQPGPNHEEDADSYENM (SEQ ID NO: 70). In some embodiments, the fusion protein comprises a sequence with at least 90% sequence identity to

(SEQ ID NO: 71) EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMG WINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFS GSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKSGGGG SGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAA ITSYEKSDGVYTGLSTRNQETYETLKHEKPPQKKVAKKPTNKAPHPKQE PQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ or (SEQ ID NO: 72) EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMG WINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFS GSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKSGGGG SGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKA AITSYEKSDGVYTGLSTRNQETYETLKHEKPPQKKVAKKPTNKAPHPKQ EPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ.

In some embodiments, the fusion protein is a transmembrane protein, an intracellular protein or an intracellular protein. In one embodiment the fusion protein is directed to enhancing the function of an immune cell, e.g., a myeloid cell, selected from monocyte, macrophages dendritic cells or precursors thereof. In one embodiment the fusion protein augments a cellular function of an immune cell, such as phagocytosis. The disclosure is not limited by the transgenes that can be expressed using the methods and compositions described. The transgenes indicated in this section are exemplary.

Provided herein are exemplary transgene candidates, for stable integration into the genome of a phagocytic cell. In one embodiment the transgene is a recombinant nucleic acid encoding a phagocytic receptor (PR) fusion protein (CFP). The recombinant nucleic acid has a PR subunit comprising: (i) a transmembrane domain, and (ii) an intracellular domain comprising a phagocytic receptor intracellular signaling domain; and an extracellular antigen binding domain specific to an antigen of a target cell; wherein the transmembrane domain and the extracellular antigen binding domain are operatively linked such that antigen binding to the target by the extracellular antigen binding domain of the fused receptor activated in the intracellular signaling domain of the phagocytic receptor. In some embodiments, the recombinant nucleic acid encodes a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is a chimeric antigen receptor (phagocytosis) (CAR-P). In some embodiments, the fusion protein is a recombinant protein for locking anti-phagocytic signals. In some embodiments, the fusion protein is a phagocytosis enhancing chimeric protein. In some embodiments, the chimeric protein has intracellular domains comprising active phagocytosis signal transduction domains. In some embodiments, the chimeric protein enhances the phagocytic potential by enhancing the inflammatory potential of the phagocytic cell in which it expresses. In some embodiments, the transgene is designed to express a chimeric protein which is activated by contact with an antigen in a target cell, whereupon the phagocytic cell phagocytoses the target cell and kills the target cell.

The terms “spacer” or “linker” as used in reference to a fusion protein refers to a peptide sequence that joins the protein domains of a fusion protein. Generally, a spacer has no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins or RNA sequences. However, in some embodiments, the constituent amino acids of a spacer can be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule. Suitable linkers for use in an embodiment of the present disclosure are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. The linker is used to separate two antigenic peptides by a distance sufficient to ensure that, in some embodiments, each antigenic peptide properly folds. Exemplary peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure. Typical amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other near neutral amino acids, such as Thr and Ala, also can be used in the linker sequence.

The various exemplary proteins encoded by a transgene that can be expressed for enhancing the immune potential of a phagocytic cell are described below. This is not an exhaustive list but serves as an exemplary list for transgene design within the scope of the present disclosure.

In some embodiments, the PSP subunit comprises a transmembrane (TM) domain of a phagocytic receptor.

In some embodiments, the PSP subunit comprises an ICD domain of a phagocytic receptor.

In some embodiments, the ICD encoded by the recombinant nucleic acid comprises a domain selected from the group consisting of lectin, dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor.

In some embodiments, the ICD comprises the signaling domain derived from any one or more of: lectin, dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO (Macrophage Receptor with Collagenous Structure, aliases: SRA6, SCARA2), CD36 (Thrombospondin receptor, aliases: Scavenger Receptor class B, member 3), CD163 (Scavenger receptor, cysteine rich-type 1), MSR1, SCARA3, COLEC12 (aliases: Scavenger Receptor With C-Type Lectin, SCARA4, or Collectin 12), SCARA5, SCARB1, SCARB2, CD68 (SCARD, microsialin), OLR1 (Oxidized Low Density Lipoprotein Receptor 1, LOX1, or C-Type Lectin Domain Family 8 Member A), SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169 (aliases, Sialoadhesin receptor, SIGLEC1).

In some embodiments, the recombinant nucleic acid encodes, for example, an intracellular domain of human MARCO. The PSR subunit comprises an intracellular domain having a 44 amino acid ICD of human MARCO having an amino acid sequence: MRNKKILKEDELLSETQQAAFHQIAMEPFEINVPKPKRRNGVNF (SEQ ID NO: 73). In some embodiments, the PSR subunit comprises a variant which is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the intracellular domain of MARCO.

In some embodiments, for example, the PSR (phagocytic scavenger receptor) comprises a transmembrane region of human MARCO.

In some embodiments, the recombinant nucleic acid encodes an intracellular domain of human SRA1. The PSR subunit comprises an intracellular domain having a 50 amino acid ICD of human SRA1 having an amino acid sequence: MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPKNSPSLQEKLKSFK (SEQ ID NO: 74). In some embodiments, the PSR subunit comprises a variant which is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the intracellular domain of human SRA1. The intracellular region of SRA has a phosphorylation site.

In some embodiments, the PSR comprises a transmembrane region of human SRA 1.

In some embodiments, for example, the recombinant nucleic acid comprises an intracellular domain of CD36. In some embodiments, the recombinant nucleic acid comprises a TM domain of CD36. Naturally occurring full length CD36 has two TM domains and two short intracellular domains, and an extracellular domain of CD36 binds to oxidized LDL. Both of the intracellular domains contain pairs of cysteines that are fatty acid acylated. It lacks known signaling domains (e.g. kinase, phosphatase, g-protein binding, or scaffolding domains). N-terminal cytoplasmic domain is extremely short (5-7 amino acid residues) and is closely associated with the internal leaflet of the plasma membrane. The carboxy-terminal domain contains 13 amino acids, containing a CXCXSK motif homologous to a region in the intracellular domain of CD4 and CD8 that is known to interact with signaling molecules. The intracellular domain of CD36 is capable of assembling a signaling complex that activates lyn kinases, MAP kinases and Focal Adhesion Kinases (FAK), and inactivation of src homology 2-containing phosphotyrosine phosphatase (SHP-2). Members of the guanine nucleotide exchange factors (GEFs) have been identified as potential key signaling intermediates.

In some embodiments, the recombinant nucleic acid encodes for example, an intracellular domain of human SCARA3. In some embodiments, the PSR subunit comprises a variant which is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the intracellular domain of human SCARA3. In some embodiments, the PSR comprises the TM domain of SCARA3. In some embodiments, the TM domains are about 20-30 amino acids long.

Scavenger receptors may occur as homo or hetero dimers. MARCO, for example occurs as a homo trimer.

In some embodiments, the TM domain or the ICD domain of the PSP is not derived from FcR, Megf10, Bai1 or MerTK. In some embodiments, the ICD of the PSR does not comprise a CD3 zeta intracellular domain.

In some embodiments, the intracellular domain and transmembrane domains are derived from FcR beta.

In one aspect the recombinant nucleic acid encodes a chimeric antigenic receptor for enhanced phagocytosis (CAR-P), which is a phagocytic scavenger receptor (PSR) fusion protein (CFP) comprising: (a) an extracellular domain comprising an extracellular antigen binding domain specific to an antigen of a target cell, (b) a transmembrane domain, and (c) a recombinant PSR intracellular signaling domain, wherein the recombinant PSR intracellular signaling domain comprises a first portion derived from a phagocytic and a second portion derived from non-phagocytic receptor.

In some embodiments, the second portion is not a PI3K recruitment domain. In some embodiments, the second portion is a PI3K recruitment domain.

The second portion derived from non-phagocytic receptor may comprise an intracellular signaling domain that enhances phagocytosis, and/or inflammatory potential of the engineered phagocytic cells expressing the recombinant nucleic acid. In some embodiments, the second portion derived from non-phagocytic receptor comprises more than one intracellular domain (ICD). In some embodiments, the second portion derived from non-phagocytic receptor comprises a second ICD. In some embodiments, the second portion derived from non-phagocytic receptor comprises a second and a third ICD. In some embodiments, the second portion derived from non-phagocytic receptor comprises a second, a third and a fourth ICD, wherein the second portion is encoded by the recombinant nucleic acid. The respective second portions comprising a second, or third or fourth ICD derived from non-phagocytic receptor are described as follows.

Chimeric Antigen Receptors for Enhancing Intracellular Signaling and Inflammation Activation

In one aspect, the recombinant nucleic acid encodes a second intracellular domain in addition to the phagocytic ICD, which confers capability of potent pro-inflammatory immune activation, such as when macrophages engage in fighting infection. The second intracellular domain (second ICD) is fused to the cytoplasmic terminus of the first phagocytic ICD. The second intracellular domain provides a second signal is necessary to trigger inflammasomes and pro-inflammatory signals. Nod-like receptors (NLRs) are a subset of receptors that are activated in innate immune response, and oligomerize to form multi-protein complexes that serve as platforms to recruit proinflammatory caspases and induce their cleavage and activation. This leads to direct activation of ROS, and often result in a violent cell death known as pyroptosis. There are four inflammasome complexes, NLRP1m, NLRP3, IPAF and AIM2.

The tumor microenvironment (TME) constitutes an immunosuppressive environment. Influence of IL-10, glucocorticoid hormones, apoptotic cells, and immune complexes can interfere with innate immune cell function. Immune cells, including phagocytic cells settle into a tolerogenic phenotype. In macrophages, this phenotype, commonly known as the M2 phenotype is distinct from the M1 phenotype, where the macrophages are potent and capable of killing pathogens. Macrophages exposed to LPS or IFN-gamma, for example, can polarize towards an M1 phenotype, whereas macrophages exposed to IL-4 or IL-13 will polarize towards an M2 phenotype. LPS or IFN-gamma can interact with Toll-like receptor 4 (TLR4) on the surface of macrophages inducing the Trif and MyD88 pathways, inducing the activation of transcription factors IRF3, AP-1, and NFKB and thus activating TNFs genes, interferon genes, CXCL10, NOS2, IL-12, etc., which are necessary in a pro-inflammatory M1 macrophage response. Similarly, IL-4 and IL-13 bind to IL-4R, activation the Jak/Stat6 pathway, which regulates the expression of CCL17, ARG1, IRF4, IL-10, SOCS3, etc., which are genes associated with an anti-inflammatory response (M2 response). Expression of CD14, CD80, D206 and low expression of CD163 are indicators of macrophage polarization towards the M1 phenotype.

In some embodiments, the recombinant nucleic acid encodes one or more additional intracellular domains, comprising a cytoplasmic domain for inflammatory response. In some embodiments, expression of the recombinant nucleic acid encoding the phagocytic receptor (PR) fusion protein (CFP) comprising the cytoplasmic domain for inflammatory response in the engineered macrophages confers potent pro-inflammatory response similar to the M1 phenotype.

In some embodiments, the cytoplasmic domain for inflammatory response can be the signal transducing domains or regions of TLR3, 4, 9, MYD88, TRIF, RIG-1, MDAS, CD40, IFN receptor, NLRP-1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, CD40.

In some embodiments, the expression of the recombinant nucleic acid encoding the phagocytic scavenger receptor (PSR) fusion protein (CFP) comprises a pro-inflammatory cytoplasmic domain for activation of IL-1 signaling cascade.

In some embodiments, the cytoplasmic portion of the chimeric receptor (for example, phagocytic receptor (PR) fusion protein (CFP)) comprises a cytoplasmic domain from a toll-like receptor, such as the intracellular signaling domains of toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), toll-like receptor 7 (TLR7), toll-like receptor 8 (TLR8), toll-like receptor 9 (TLR9). In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from interleukin-1 receptor-associated kinase 1 (IRAK1)._In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from differentiation primary response protein (MYD88). In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from myelin and lymphocyte protein (MAL). In some embodiments, the cytoplasmic portion of the chimeric receptor comprises a suitable region from retinoic acid inducible gene (RIG-1).

In some embodiments, the transmembrane domain of the PSR comprises the transmembrane domain of any one of MYD88, TLR3, TLR4, TLR7, TLR8, TLR9, MAL, IRAK1, proteins.

In some embodiments, the recombinant PSR intracellular signaling domain comprises a first portion derived from a phagocytic and a second portion derived from non-phagocytic receptor wherein the second portion derived from non-phagocytic receptor comprises a phosphorylation site. In some embodiments, the phosphorylation site comprises amino acid sequences suitable for an autophosphorylation site. In some embodiments, the phosphorylation site comprises amino acid sequences suitable phosphorylation by Src family kinases. In some embodiments, the phosphorylation site comprises amino acid sequences, which upon phosphorylation are capable of binding to SH2 domains in a kinase. In some embodiments, a receptor tyrosine kinase domain is fused at the cytoplasmic end of the CFP in addition to the first cytoplasmic portion. In some embodiments, the phosphorylation is a tyrosine phosphorylation.

In some embodiments, the second intracellular domain is an Immune receptor Tyrosine Activation Motif (ITAM). The ITAM motif is present in mammalian α and β immunoglobulin proteins, TCR γ receptors, FCR γ receptors subunits, CD3 chains receptors and NFAT activation molecule.

In some embodiments, the CFP intracellular domain comprises one ITAM motif. In some embodiments, the CFP intracellular domain comprises more than one ITAM motifs. In some embodiments, the CFP intracellular domain comprises two or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises three or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises four or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises five or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises six or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises seven or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises eight or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises nine or more ITAM motifs. In some embodiments, the CFP intracellular domain comprises ten or more ITAM motifs.

In some embodiments, one or more domains in the first phagocytic ICD comprises a mutation.

In some embodiments, one or more domains in the second ICD comprises a mutation to enhance a kinase binding domain, to generate a phosphorylation site, to generate an SH2 docking site or a combination thereof.

Co-Expression of an Inflammatory Gene

In one aspect, the recombinant nucleic acid comprises a coding sequence for a pro-inflammatory gene, which is co-expressed with the CFP in the engineered cell. In some embodiments, the pro-inflammatory gene is a cytokine. Examples include but not limited to TNF-α, IL-1α, IL-1β, IL-6, CSF, GMCSF, or IL-12 or interferons.

The recombinant nucleic acid encoding the proinflammatory gene can be monocistronic, wherein the two coding sequences for (a) the PSP and (b) the proinflammatory gene are post-transcriptionally or post-translationally cleaved for independent expression.

In some embodiments, the two coding sequences comprise a self-cleavage domain, encoding a P2A sequence, for example.

In some embodiments, the two coding regions are separated by an IRES site.

In some embodiments, the two coding sequences are encoded by a bicistronic genetic element. The coding regions for (a) the PSP and (b) the proinflammatory gene can be unidirectional, where each is under a separate regulatory control. In some embodiments, the coding regions for both are bidirectional and drive in opposite directions. Each coding sequence is under a separate regulatory control.

Co-expression of the proinflammatory gene is designed to confer strong inflammatory stimulation of the macrophage and activate the surrounding tissue for inflammation.

Integrin Activation Domains

Cell-cell and cell-substratum adhesion is mediated by the binding of integrin extracellular domains to diverse protein ligands; however, cellular control of these adhesive interactions and their translation into dynamic cellular responses, such as cell spreading or migration, requires the integrin cytoplasmic tails. These short tails bind to intracellular ligands that connect the receptors to signaling pathways and cytoskeletal networks (Calderwood DA, 2004, Integrin Activation, Journal of Cell Science 117, 657-666). Integrins are heterodimeric adhesion receptors formed by the non-covalent association of α and β subunits. Each subunit is a type I transmembrane glycoprotein that has relatively large extracellular domains and, with the exception of the β4 subunit, a short cytoplasmic tail. Individual integrin family members have the ability to recognize multiple ligands. Integrins can bind to a large number of extracellular matrix proteins (bone matrix proteins, collagens, fibronectins, fibrinogen, laminins, thrombospondins, vitronectin, and von Willebrand factor), reflecting the primary function of integrins in cell adhesion to extracellular matrices. Many “counter-receptors” are ligands, reflecting the role of integrins in mediating cell-cell interactions. Integrins undergo conformational changes to increase ligand affinity.

The Integrin β₂ subfamily consists of four different integrin receptors, α_(M)β₂ (CD11b/CD18, Mac-1, CR3, Mo-1), α_(L)β₂ (CD11a/CD18, LFA-1), α_(X)β₂ (CD11c/CD18), and α_(D)β₂ (CD11d/CD18). These leukocyte integrins are involved in virtually every aspect of leukocyte function, including the immune response, adhesion to and transmigration through the endothelium, phagocytosis of pathogens, and leukocyte activation.

The α subunits of all β₂ integrins contain an inserted region of ˜200 amino acids, termed the I or A domain. Highly conserved I domains are found in several other integrin α subunits and other proteins, such as certain coagulation and complement proteins. I domains mediate protein-protein interactions, and in integrins, they are integrally involved in the binding of protein ligands. Although the I domains dominate the ligand binding functions of their integrins, other regions of the α subunits do influence ligand recognition. As examples, in α_(M)β₂ a mAb (OKM1) recognizing an epitope outside the I domain but in the α_(M) subunit inhibits ligand binding; and the EF-hand regions in α_(L)β₂ and α₂β₁, integrins with I domains in their α subunits, contribute to ligand recognition. The α_(M) subunit, and perhaps other α subunits, contains a lectin-like domain, which is involved in engagement of non-protein ligands, and occupancy may modulate the function of the I domain.

As integrins lack enzymatic activity, signaling is instead induced by the assembly of signaling complexes on the cytoplasmic face of the plasma membrane. Formation of these complexes is achieved in two ways; first, by receptor clustering, which increases the avidity of molecular interactions thereby increasing the on-rate of binding of effector molecules, and second, by induction of conformational changes in receptors that creates or exposes effector binding sites. Within the ECM, integrins have the ability to bind fibronectin, laminins, collagens, tenascin, vitronectin and thrombospondin. Clusters of integrin/ECM interactions form focal adhesions, concentrating cytoskeletal components and signaling molecules within the cell. The cytoplasmic tail of integrins serve as a binding site for α-actinin and talin which then recruit vinculin, a protein involved in anchoring F-actin to the membrane. Talin is activated by kinases such as protein kinase C (PKCα).

Integrins are activated by selectins. Leucocytes express L-selectin, activated platelets express P-selectin, and activated endothelial cells express E- and P-selectin. P-selectin-mediated adhesion enables chemokine- or platelet-activating factor-triggered activation of β2 integrins, which stabilizes adhesion. It also facilitates release of chemokines from adherent leucocytes. The cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-associated factor 1. After binding of P-selectin, Src kinases phosphorylated Nef-associated factor 1, which recruit the phosphoinositide-3-OH kinase p85-p110δ heterodimer and result in activation of leukocyte integrins. E-selectin ligands transduce signals that also affect β2 integrin function. Selectins trigger activation of Src family kinases. SFKs activated by selectin engagement phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of DAP 12 and FcRγ. In some respects, CD44 is sufficient to transduce signals from E-selectin. CD44 triggers the inside-out signaling of integrins. A final common step in integrin activation is binding of talin to the cytoplasmic tail of the β subunit. Kindlins, another group of cytoplasmic adaptors, bind to a different region of integrin β tails. Kindlins increase the clustering of talin-activated integrins. Kindlins are responsive to selectin signaling, however, kindlins are found mostly in hematopoietic cells, such as neutrophils. Selectin signaling as well as signaling upon integrin activation by chemokines components have shared components, including SFKs, Syk, and SLP-76.

In some embodiments, the intracellular domain of the recombinant PSR fusion protein comprises an integrin activation domain. The integrin activation domain comprises an intracellular domain of a selectin, for example, a P-selectin, L-selectin or E-selectin.

In some embodiments, the intracellular domain of the recombinant PSR fusion protein comprises an integrin activation domain of laminin.

In some embodiments, the intracellular domain of the recombinant PSR fusion protein comprises an integrin activation domain for activation of Talin.

In some embodiments, the intracellular domain of the recombinant PSR fusion protein comprises an integrin activation domain fused to the cytoplasmic end of the phagocytic receptor ICD domain.

Chimeric Receptor for Enhancing Antigen Cross Presentation

In some embodiments, the recombinant nucleic acid encodes a domain capable of enabling cross presentation of antigens. In general, MHC class I molecules present self- or pathogen-derived antigens that are synthesized within the cell, whereas exogenous antigens derived via endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. MHC I-restricted presentation of endogenous antigens, in which peptides are generated by the proteasome. However, in some cases, DC can process exogenous antigens into the MHC-I pathway for presentation to CD8+ T cells. This is referred to as cross presentation of antigens. Soluble or exogenous antigenic components may get degraded by lysosomal proteases in the vacuoles and cross presented by DCs, instead of following the endocytotic pathway. In some instances, chaperones, such as heat shock protein 90 (Hsp90) have shown to help cross present antigens by certain APCs. HSP-peptide complexes are known to be internalized by a distinct group of receptors compared to free polypeptides. These receptors are from the scavenger receptor families and included LOX-1, SREC-I/SCARF-I, and FEEL1/Stabilin-1. Both SREC-I and LOX-1 have been shown to mediate the cross presentation of molecular chaperone bound antigens and lead to activation of CD8⁺ T lymphocytes.

SREC-1 (scavenger receptor expressed by endothelial cells) has no significant homology to other types of scavenger receptors but has unique domain structures. It contains 10 repeats of EGF-like cysteine-rich motifs in the extracellular domain. Recently, the structure of SREC-I was shown to be similar to that of a transmembrane protein with 16 EGF-like repeats encoded by the Caenorhabditis elegans gene ced-I, which functions as a cell surface phagocytic receptor that recognizes apoptotic cells.

Cross presentation of cancer antigens through the Class-I MHC pathway results in enhanced CD8+ T cell response, which is associated with cytotoxicity and therefore beneficial in tumor regression. In some embodiments, the intracellular domain of the CFP comprises a SREC1 intracellular domain. In some embodiments, the intracellular domain of the CFP comprises a SRECII intracellular domain.

In some embodiments, the PSR subunit comprises: an intracellular domain comprising a PSR intracellular signaling domain from SREC1 or SRECII.

In some embodiments, the PSR subunit comprises: (i) a transmembrane domain, and (ii) an intracellular domain comprising a PSR intracellular signaling domain from SREC1 or SRECII.

In some embodiments, the PSR subunit comprises: (i) a transmembrane domain, (ii) an intracellular domain comprising a PSR intracellular signaling domain, and (iii) an extracellular domain from SREC1 or SRECII.

Transmembrane Domain of a CFP Fusion Protein

In some embodiments, the TM encoded by the recombinant nucleic acid comprises a domain of a scavenger receptor (SR). In some embodiments, the TM can be the TM domain of or derived from any one or more of: lectin, dectin 1, mannose receptor (CD206), SRA1, MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, SRCRB4D, SSC5D, and CD169.

In some embodiments, the TM domains are about 20-30 amino acids long. TM domains of SRs are about 20-30 amino acids long.

The TM domain or the ICD domain of the PSP is not derived from Megf10, Bai1 or MerTK. The ICD of the PSR does not comprise a CD3 zeta intracellular domain.

In some embodiments, the TM is derived from the same phagocytic receptor as the ICD.

In some embodiments, the TM region is derived from a plasma membrane protein. The TM can be selected from an Fc receptor (FcR). In some embodiments, nucleic acid sequence encoding domains from specific FcRs are used for cell-specific expression of a recombinant construct. An FCR-alpha region comprising the TM domain may be used for macrophage specific expression of the construct. FcRβ recombinant protein expresses in mast cells.

In some embodiments, the CFP comprises the TM of an FCR-beta (FcRβ).

In some embodiments, the CFP comprises both the FcRβ TM and ICD domains.

In some embodiments, the TM domain is derived from CD8.

In some embodiments, the TM is derived from CD2.

In some embodiments, the TM is derived from FCR alpha.

Extracellular Domain of a CFP Fusion Protein

The extracellular domain comprises an antigen binding domain that binds to one or more target antigens on a target cell. The target binding domain is specific for the target. The extracellular domain can include an antibody or an antigen-binding domain selected from intrabodies, peptibodies, nanobodies, single domain antibodies. SMIPs, and multispecific antibodies.

In some embodiments, the extracellular domain includes a Fab binding domain. In yet other such embodiments, the extracellular domain includes a scFv.

In some embodiments, the chimeric antigen receptor comprises an extracellular antigen binding domain is derived from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a VNAR domain, and a VHH domain, a bispecific antibody, a diabody, or a functional fragment of any thereof. In some embodiments, the antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a VNAR domain, and a VHH domain, a bispecific antibody, a diabody, or a functional fragment of any thereof specifically bind to one or more antigens.

In some embodiments, the antigens are cancer antigens, and the target cell is a target cancer cell. In some embodiments, the antigen for a target cancer cell is selected from the group consisting of CD3, CD4, CD5, CD7, CD19, CCR2, CCR4, CD30, CD37, TCRB1/2, TCR □ □, TCR □ □. CD22, HER2 (ERBB2/neu), Mesothelin, PSCA, CD123, CD30, CD171, CD138, CS-1, CLECL1, CD33, CD79b, EGFRvIII, GD2, GD3, BCMA, PSMA, ROR1, FLT3, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3 (CD276), KIT (CD 117), CD213A2, IL-1 IRa, PRSS21, VEGFR2, CD24, MUC-16, PDGFR-beta, SSEA-4, CD20, MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, FAP, EphA2, GM3, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CD97, CD179a, ALK, and IGLL1.

Various cancer antigen targets can be selected from cancer antigens known to one of skill in the art. Depending on the cancer and the cell type involved cancer antigens are mutated native proteins. The antigen binding domains are screened for specificity towards mutated/cancer antigens and not the native antigens.

In some embodiments, for example, the cancer antigen for a target cancer cell can be one or more of the mutated/cancer antigens: MUC16, CCAT2, CTAG1A, CTAG1B, MAGE A1, MAGEA2, MAGEA3, MAGE A4, MAGEA6, PRAME, PCA3, MAGE C1, MAGEC2, MAGED2, AFP, MAGEA8, MAGE9, MAGEA11, MAGEA12, IL13RA2, PLAC1, SDCCAG8, LSP1, CT45A1, CT45A2, CT45A3, CT45A5, CT45A6, CT45A8, CT45A10, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A8, CT47A9, CT47A10, CT47A11, CT47A12, CT47B1, SAGE1, and CT55.

In some embodiments, for example, the cancer antigen for a target cancer cell can be one or more of the mutated/cancer antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30, CD45, CD56, where the cancer is a T cell lymphoma.

In some embodiments, for example, the cancer antigen for a target cancer cell can be one or more of the mutated/cancer antigens: IDH1, ATRX, PRL3, or ETBR, where the cancer is a glioblastoma.

In some embodiments, for example, the cancer antigen for a target cancer cell can be one or more of the mutated/cancer antigens: CA125, beta-hCG, urinary gonadotropin fragment, AFP, CEA, SCC, inhibin or extradiol, where the cancer is ovarian cancer.

In some embodiments, the cancer antigen for a target cancer cell may be HER2.

In some embodiments, the cancer antigen for a target cancer cell may be EGFR Variant III.

In some embodiments, the cancer antigen for a target cancer cell may be CD19.

In some embodiments, the SR subunit region comprises an extracellular domain (ECD) of the scavenger receptor. In some embodiments, the ECD of the scavenger receptor comprises an ECD domain of the SR comprising the ICD and the TM domains. In some embodiments, the SR-ECD contributes to the binding of the phagocyte to the target cell, and in turn is activated, and activates the phagocytosis of the target cell.

In some embodiments, the PSR domain optionally comprises the ECD domain or portion thereof of the respective scavenger receptor the ICD and TM domains of which is incorporated in the PSR. Therefore, in some embodiments, In some embodiments, the ECD encoded by the recombinant nucleic acid comprises a domain selected from the group consisting of lectin, dectin 1, mannose receptor (CD206), scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4/80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), and CD169 receptor. The extracellular domains of most macrophage scavenger receptors contain scavenger receptors with a broad binding specificity that may be used to discriminate between self and non-self in the nonspecific antibody-independent recognition of foreign substances. The type I and II class A scavenger receptors (SR-AI1 and SR-AII) are trimeric membrane glycoproteins with a small NH2-terminal intracellular domain, and an extracellular portion containing a short spacer domain, an a-helical coiled-coil domain, and a triple-helical collagenous domain. The type I receptor additionally contains a cysteine-rich COOH-terminal (SRCR) domain. These receptors are present in macrophages in diverse tissues throughout the body and exhibit an unusually broad ligand binding specificity. They bind a wide variety of polyanions, including chemically modified proteins, such as modified LDL, and they have been implicated in cholesterol deposition during atherogenesis. They may also play a role in cell adhesion processes in macrophage-associated host defense and inflammatory conditions.

In some embodiments, the SR ECD is designed to bind to pro-apoptotic cells. In some embodiments, the scavenger receptor ECD comprises a binding domain for a cell surface molecule of a cancer cell or an infected cell.

In some embodiments, the extracellular domain of the PR subunit is linked by a linker to a target cell binding domain, such as an antibody or part thereof, specific for a cancer antigen.

In some embodiments, the extracellular antigen binding domain comprises one antigen binding domain. In some embodiments, the extracellular antigen binding domain comprises more than one binding domain. In some embodiments, the binding domain is an scFv. In some embodiments, the binding domain is an single domain antibody (sdAb). In some embodiments, the binding domain is fused to the recombinant PR at the extracellular domain. In some embodiments, the binding domain (e.g., scFv) and the extracellular domain of the PR are linked via a linker.

In some embodiments, the ECD antigen binding domain can bind to an intracellular antigen. In some embodiments, the intracellular antigen is a cancer antigen.

In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 1000 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 500 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 450 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 400 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 350 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 250 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 200 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity of less than 100 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity ranging between than 200 nM to 1000 nM. In some embodiments, the extracellular antigen binding domain binds to the target ligand with an affinity ranging between than 300 nM to 1.5 mM. In some embodiments, the antigen binding domain binds to the target ligand with an affinity >200 nM, >300 nM or >500 nM.

Peptide Linker

In some embodiments, the extracellular antigen binding domains, scfvs are linked to the TM domain or other extracellular domains by a linker. In some embodiments, where there are more than one scfv at the extracellular antigen binding domain the more than scfvs are linked with each other by linkers.

In some embodiments, the linkers are flexible. In some embodiments, the linkers comprise a hinge region. Linkers are usually short peptide sequences. In some embodiments, the linkers are stretches of Glycine and one or more Serine residues. Other amino acids preferred for short peptide linkers include but are not limited to threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), and alanine (Ala) arginine (Arg), phenylalanine (Phe), glutamic acid (Glu). Of these Pro, Thr, and Gln are frequently used amino acids for natural linkers. Pro is a unique amino acid with a cyclic side chain which causes a very restricted conformation. Pro-rich sequences are used as interdomain linkers, including the linker between the lipoyl and E3 binding domain in pyruvate dehydrogenase (GA₂PA₃PAKQEA₃PAPA₂KAEAPA₃PA₂KA) (SEQ ID NO: 75). For the purpose of the disclosure, the empirical linkers may be flexible linkers, rigid linkers, and cleavable linkers. Sequences such as (G4S)_(X) (SEQ ID NO: 76) (where x is multiple copies of the moiety, designated as 1, 2, 3, 4, and so on) comprise a flexible linker sequence. Other flexible sequences used herein include several repeats of glycine, e.g., (Gly)6 (SEQ ID NO: 77) or (Gly)8 (SEQ ID NO: 78). On the other hand, a rigid linker may be used, for example, a linker (EAAAK)_(X) (SEQ ID NO: 79), where x is an integer, 1, 2, 3, 4 etc. gives rise to a rigid linker.

In some embodiments, the linker comprises at least 2, or at least 3 amino acids. In some embodiments, the linker comprises 4 amino acids. In some embodiments, the linker comprises 5 amino acids. In some embodiments, the linker comprises 6 amino acids. In some embodiments, the linker comprises 7 amino acids. In some embodiments, the linker comprises 8 amino acids. In some embodiments, the linker comprises 9 amino acids. In some embodiments, the linker comprises 8 amino acids. In some embodiments, the linker comprises 10 amino acids. In some embodiments, the linker comprises 11 amino acids. In some embodiments, the linker comprises 12 amino acids. In some embodiments, the linker comprises 13 amino acids. In some embodiments, the linker comprises 14 amino acids. In some embodiments, the linker comprises 15 amino acids. In some embodiments, the linker comprises 16 amino acids. In some embodiments, the linker comprises 17 amino acids. In some embodiments, the linker comprises 18 amino acids. In some embodiments, the linker comprises 19 amino acids. In some embodiments, the linker comprises 20 amino acids.

As contemplated herein, any suitable ECD, TM or ICD domain can be cloned interchangeably in the suitable portion of any one of the CARP receptors described in the disclosure to obtain a protein with enhanced phagocytosis compared to an endogenous receptor.

Characteristics of the Fusion Proteins:

The CFP can structurally incorporate into the cell membrane of the cell in which it is expressed. Specific leader sequences in the nucleic acid construct, such as the signal peptide can be used to direct plasma membrane expression of the encoded protein. The transmembrane domain encoded by the construct can incorporate the expressed protein in the plasma membrane of the cell.

In some embodiments, the transmembrane domain comprises a TM domain of an FcRalpha receptor, which dimerizes with endogenous FcR-gamma receptors in the macrophages, ensuring macrophage specific expression.

The CFP can render the cell that expresses it as potently phagocytic. When the recombinant nucleic acid encoding the CFP is expressed in a cell, the cell can exhibit an increased phagocytosis of a target cell having the antigen of a target cell, compared to a cell not expressing the recombinant nucleic acid. When the recombinant nucleic acid is expressed in a cell, the cell can exhibit an increased phagocytosis of a target cell having the antigen of a target cell, compared to a cell not expressing the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid when expressed in a cell, the cell exhibits at least 2-fold increased phagocytosis of a target cell having the antigen of a target cell, compared to a cell not expressing the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid when expressed in a cell, the cell exhibits at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold 30-fold or at least 5-fold increased phagocytosis of a target cell having the antigen of a target cell, compared to a cell not expressing the recombinant nucleic acid.

In some embodiments, expression of SIRP-ΔICD enhances phagocytosis of the cell expressing it by 1.1 fold or more, 1.2 fold or more, 1.3 fold or more, q.4 fold or more, 1.5 fold or more, by 1.6 fold or more, 1.7 fold or more, 1.8 fold or more, 1.9 fold or more, 2 fold or more, 3 fold or more, 4 fold or more, 5 fold or more, 8 fold or more, 10 fold or more, 15 fold or more, 20 fold or more, 30 fold or more, 40 fold or more, 50 fold or more, 60 fold or more, 70 fold or more 80 fold or more, 90 fold or more, 100 fold or more, compared to a cell not expressing SIRP-ΔICD.

In some embodiments, the cells co-expressing SIRP-ΔICD and a CFP encoding a phagocytic receptor as described herein exhibits an augmented phagocytosis compared to a cell that does not express either of the proteins. In some embodiments, co-expressing SIRP-ΔICD and a CFP encoding a phagocytic receptor as described herein exhibits more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, more than 9-fold, more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold, more than 60-fold, more than 70-fold, more than 80-fold, more than 90-fold, more than 100-fold, or more than 150-fold or more than 200-fold increase in phagocytic potential (measured in fold change of phagocytic index) compared to a cell that does not express either the SIRP-ΔICD or the CFP encoding a phagocytic receptor.

In some embodiments, expression of the any one of a CFP expressing a CD47 blocking extracellular domain of SIRPα and an intracellular domain of a phagocytic receptor augments phagocytic activity of a cell expressing it by at least 1.5 fold or more, 1.6 fold or more, 1.7 fold or more, 1.8 fold or more, 1.9 fold or more, 2 fold or more, 3 fold or more, 4 fold or more, 5 fold or more, 8 fold or more, 10 fold or more, 15 fold or more, 20 fold or more, 30 fold or more, 40 fold or more, 50 fold or more, 60 fold or more, 70 fold or more 80 fold or more, 90 fold or more, 100 fold or more, compared to a cell not expressing the CFP, or compared to a cell expressing SIRP-ΔICD.

In some embodiments, the enhancement in phagocytosis of target cells by a cell expressing either SIRP-ΔICD is highly increased compared to a phagocytic cell not expressing SIRP-ΔICD.

In some embodiments, the enhancement in phagocytosis of target cells by a cell expressing a CFP comprising a CD47 blocking extracellular domain of SIRPα and an intracellular domain of a phagocytic receptor is highly increased compared to a control phagocytic cell not expressing the fusion protein or a control phagocytic cell expressing the SIRP-ΔICD.

In some embodiments, when the recombinant nucleic acid described herein is expressed in a cell, the cell exhibits an increased cytokine production. The cytokine can comprise any one of: IL-1, IL-6, IL-12, IL-23, TNF, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27 and interferons.

In some embodiments, when the recombinant nucleic acid described herein is expressed in a cell, the cell exhibits an increased cell migration.

In some embodiments, when the recombinant nucleic acid described herein is expressed in a cell, the cell exhibits an increased immune activity. In some embodiments, when the recombinant nucleic acid is expressed in a cell, the cell exhibits an increased expression of MHC II. In some embodiments, when the recombinant nucleic acid is expressed in a cell, the cell exhibits an increased expression of CD80. In some embodiments, when the recombinant nucleic acid is expressed in a cell, the cell exhibits an increased expression of CD86. In some embodiments, when the recombinant nucleic acid is expressed in a cell, the cell exhibits an increased iNOS production.

In some embodiments, when the recombinant nucleic acid is expressed in a cell, the cell exhibits decreased trogocytosis of a target cell expressing the antigen of a target cell compared to a cell not expressing the recombinant nucleic acid.

In embodiments, the chimeric receptors may be glycosylated, pegylated, and/or otherwise post-translationally modified. In further embodiments, glycosylation, pegylation, and/or other posttranslational modifications may occur in vivo or in vitro and/or may be performed using chemical techniques. In additional embodiments, any glycosylation, pegylation and/or other posttranslational modifications may be N-linked or O-linked. In embodiments any one of the chimeric receptors may be enzymatically or functionally active such that, when the extracellular domain is bound by a ligand, a signal is transduced to polarize a macrophage.

In some embodiments, the chimeric fusion protein (CFP) comprises an extracellular domain (ECD) targeted to bind to CD5 (CD5 binding domain), for example, comprising a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the chimeric CFP comprises a CD5 binding heavy chain variable domain comprising an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1. In some embodiments, the extracellular domain (ECD) targeted to bind to CD5 (CD5 binding domain) comprises a light chain variable domain (VL) having an amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the chimeric CFP comprises a CD5 binding light chain variable domain comprising an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 2.

In some embodiments, the CFP comprises an extracellular domain targeted to bind to HER2 (HER2 binding domain) having for example a heavy chain variable domain amino acid sequence as set forth in SEQ ID NO: 8 and a light chain variable domain amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the CFP comprises a HER2 binding heavy chain variable domain comprising an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 8. In some embodiments, the CFP comprises a HER2 binding light chain variable domain comprising an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 9.

In some embodiments, the CFP comprises a hinge connecting the ECD to the transmembrane (TM). In some embodiments the hinge comprises the amino acid sequence of the hinge region of a CD8 receptor. In some embodiments, the CFP may comprise a hinge having the amino acid sequence set forth in SEQ ID NO: 7 (CD8a chain hinge domain) In some embodiments, the PFP hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 7.

In some embodiments, the CFP comprises a CD8 transmembrane region, for example having an amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the CFP TM region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 6.

In some embodiments, the CFP comprises an intracellular domain having an FcR domain. In some embodiments, the CFP comprises an FcR domain intracellular domain comprises an amino acid sequence set forth in SEQ ID NO: 3, or at least a sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 3.

In some embodiments, the CFP comprises an intracellular domain having a PI3K recruitment domain. In some embodiments the PI3K recruitment domain comprises an amino sequence set forth in SEQ ID NO: 4. In some embodiments the PI3K recruitment domain comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 4.

In some embodiments, the CFP comprises an intracellular domain having a CD40 intracellular domain. In some embodiments the CD40 ICD comprises an amino sequence set forth in SEQ ID NO: 5. In some embodiments the CD40 ICD comprises an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 5.

In some embodiments, the CD5 binding domain comprises an scFv comprising: (i) a variable heavy chain (V_(H)) sequence of SEQ ID NO: 1 or with at least 90% sequence identity to SEQ ID NO: 1; and (ii) a variable light chain (V_(L)) sequence of SEQ ID NO: 2 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the CD5 binding domain comprises an scFv comprising SEQ ID NO: 33 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the HER2 binding domain comprises an scFv comprising: (i) a variable heavy chain (V_(H)) sequence of SEQ ID NO: 8 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8; and (ii) a variable light chain (V_(L)) sequence of SEQ ID NO: 9 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the CD5 binding domain comprises an scFv comprising SEQ ID NO: 32 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32. In some embodiments, the CFP further comprises an intracellular domain, wherein the intracellular domain comprises one or more intracellular signaling domains, and wherein a wild-type protein comprising the intracellular domain does not comprise the extracellular domain.

In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operatively linked to the transmembrane domain and the anti-CD5 binding domain. In some embodiments, the extracellular hinge domain comprises a sequence of SEQ ID NO: 7 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7.

In some embodiments, the CFP comprises an extracellular domain fused to a transmembrane domain of SEQ ID NO: 30 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30. In some embodiments, the CFP comprises an extracellular domain fused to a transmembrane domain of SEQ ID NO: 31 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31.

In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises a sequence of SEQ ID NO: 6 or 29 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6 or 29. In some embodiments, the transmembrane domain comprises a sequence of SEQ ID NO: 18 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a sequence of SEQ ID NO: 34 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 34. In some embodiments, the transmembrane domain comprises a sequence of SEQ ID NO: 19 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19.

In some embodiments, the CFP comprises one or more intracellular signaling domains that comprise a phagocytic signaling domain. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, FcRα, and Bai1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than Megf10, MerTk, an FcR, and Bai1. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from a receptor other than CD3. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from FcRγ, FcRα or FGRε. In some embodiments, the phagocytosis signaling domain comprises an intracellular signaling domain derived from CD3. In some embodiments, the CFP comprises an intracellular signaling domain of any one of SEQ ID NOs: 3, 20, 27 and 28 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 3, 20, 27 and 28. In some embodiments, the one or more intracellular signaling domains further comprises a proinflammatory signaling domain. In some embodiments, the proinflammatory signaling domain comprises a PI3-kinase (PI3K) recruitment domain. In some embodiments, the proinflammatory signaling domain comprises a sequence of SEQ ID NO: 4 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the proinflammatory signaling domain is derived from an intracellular signaling domain of CD40. In some embodiments, the proinflammatory signaling domain comprises a sequence of SEQ ID NO: 5 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the CFP comprises an intracellular signaling domain of SEQ ID NO: 21 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21. In some embodiments, the CFP comprises an intracellular signaling domain of SEQ ID NO: 23 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 23.

In some embodiments, the CFP comprises a sequence of SEQ ID NO: 14 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the CFP comprises a sequence of SEQ ID NO: 15 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the CFP comprises a sequence of SEQ ID NO: 16 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the CFP comprises a sequence of SEQ ID NO: 24 or with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 24. In some embodiments, the CFP comprises a sequence of SEQ ID NO:25 or with at least 70%, 75%, 80%, 85%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25.

In some embodiments, the CFP comprises: (a) an extracellular domain comprising: (i) a scFv that specifically binds CD5, and (ii) a hinge domain derived from CD8; a hinge domain derived from CD28 or at least a portion of an extracellular domain from CD68; (b) a CD8 transmembrane domain, a CD28 transmembrane domain, a CD2 transmembrane domain or a CD68 transmembrane domain; and (c) an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from FcRα, FcRγ or FcRε, and (ii) a second intracellular signaling domain: (A) comprising a PI3K recruitment domain, or (B) derived from CD40. In some embodiments, the CFP comprises as an alternative (c) to the above: an intracellular domain comprising at least two intracellular signaling domains, wherein the at least two intracellular signaling domains comprise: (i) a first intracellular signaling domain derived from a phagocytic receptor intracellular domain, and (ii) a second intracellular signaling domain derived from a scavenger receptor phagocytic receptor intracellular domain comprising: (A) comprising a PI3K recruitment domain, or (B) derived from CD40. Exemplary scavenger receptors from which an intracellular signaling domain may be derived may be found in Table 2. In some embodiments, the CFP comprises and intracellular signaling domain derived from an intracellular signaling domain of an innate immune receptor.

In some embodiments, the recombinant polynucleic acid is an mRNA. In some embodiments, the recombinant polynucleic acid is a circRNA. In some embodiments, the recombinant polynucleic acid is a viral vector. In some embodiments, the recombinant polynucleic acid is delivered via a viral vector.

In some embodiments, the myeloid cell is a CD14+ cell, a CD14+/CD16− cell, a CD14+/CD16+ cell, a CD14−/CD16+ cell, CD14−/CD16− cell, a dendritic cell, an M0 macrophage, an M2 macrophage, an M1 macrophage or a mosaic myeloid cell/macrophage/dendritic cell.

In one aspect, provided herein is a method of treating cancer in a human subject in need thereof comprising administering a pharmaceutical composition to the human subject, the pharmaceutical composition comprising: (a) a myeloid cell comprising a recombinant polynucleic acid sequence, wherein the polynucleic acid sequence comprises a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (i) an extracellular domain comprising an anti-CD5 binding domain, and (ii) a transmembrane domain operatively linked to the extracellular domain; and (b) a pharmaceutically acceptable carrier; wherein the myeloid cell expresses the CFP.

In some embodiments, upon binding of the CFP to CD5 expressed by a target cancer cell of the subject killing or phagocytosis activity of the myeloid cell is increased by greater than 20% compared to a myeloid cell not expressing the CFP. In some embodiments, growth of a tumor is inhibited in the human subject.

In some embodiments, the cancer is a CD5+ cancer. In some embodiments, the cancer is leukemia, T cell lymphoma, or B cell lymphoma. In some embodiments, the CFP comprises one or more sequences shown in Table A and/or Table B below.

TABLE A Exemplary sequences of CFPs and domains thereof SEQ ID NO PFP/Domain Sequence  1 Anti-CD5 heavy EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQA chain variable PGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAY domain LQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTV  2 Anti-CD5 light DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPG chain variable KAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDF domain GIYYCQQYDESPWTFGGGTKLEIK 33 Anti-CD5 scFv EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQA PGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAY LQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDI NSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTD YTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIK  3 FcRγ-chain LYCRRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKH intracellular EKPPQ signaling domain 20 FcRγ-chain LYCRLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHE intracellular KPPQ signaling domain 27 FcRγ-chain RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPP intracellular Q signaling domain 28 FcRγ-chain RLKIQVRKAAITSYEKSDGVYTGLSTRNQETYETLKHEKPP intracellular Q signaling domain  4 PI3K recruitment YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM domain  5 CD40 intracellular KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLH domain GCQPVTQEDGKESRISVQERQ  6 CD8α chain IYIWAPLAGTCGVLLLSLVIT transmembrane domain 29 CD8α chain IYIWAPLAGTCGVLLLSLVITLYC transmembrane domain  7 CD8α chain hinge ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSL domain RPEACRPAAGGAVHTRGLD  8 Anti-HER2 heavy DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP chain variable GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF domain ATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGS EVQLVE  9 Anti-HER2 light LVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR chain variable IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDT domain AVYYCSRWGGDGFYAMDVWGQGTLVTV 32 Anti-HER2 scFv DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF ATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGS EVQLVESSGGGGSGGGGSGGGGSLVQPGGSLRLSCAASGFN IKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAM DVWGQGTLVTV 17 GMCSF Signal MWLQSLLLLGTVACSIS peptide 18 CD28 FWVLVVVGGVLACYSLLVTVAFIIFWV transmembrane domain 34 CD2 IYLIIGICGGGSLLMVFVALLVFYIT Transmembrane domain 19 CD68 ILLPLIIGLILLGLLALVLIAFCII transmembrane domain 21 TNFR1 QRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPG intracellular FTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQG domain ADPILATALASDPIPNPLQKWEDSAHKPQSLDTDDPATLYA VVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQY SMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEAL CGPAALPPAPSLLR 22 TNFR2 PLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLE intracellular SSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPG domain GHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSE SPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDA GMKPS 23 MDA5 MSNGYSTDENFRYLISCFRARVKMYIQVEPVLDYLTFLPAE intracellular VKEQIQRTVATSGNMQAVELLLSTLEKGVWHLGWTREFVE domain ALRRTGSPLAARYMNPELTDLPSPSFENAHDEYLQLLNLLQ PTLVDKLLVRDVLDKCMEEELLTIEDRNRIAAAENNGNESG VRELLKRIVQKENWFSAFLNVLRQTGNNELVQELTGSDCSE SNAEIEN 30 CD8a chain hinge ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSL domain + RPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVITL transmembrane YC domain 31 CD8a chain hinge ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSL domain + RPEACRPAAGGAVHTRGLDIYIWAPLAGTCGVLLLSLVIT transmembrane domain 14 CD5-FcRy-PI3K MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAA SGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRA NRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDE SPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDI YIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKAAITSYEKSD GVYTGLSTRNQETYETLKHEKPPQGSGSYEDMRGILYAAPQ LRSIRGQPGPNHEEDADSYENM 15 HER2-FcRγ-PI3K MWLQSLLLLGTVACSISDIQMTQSPSSLSASVGDRVTITCRA SQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR TGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDVWGQGTLVTVSSSGGGGSGALSNSIMYFSHFVPVFLP AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDIYIWAPLAGTCGVLLLSLVITLYCRRLKIQVRKAAITSYE KSDGVYTGLSTRNQETYETLKHEKPPQGSGSYEDMRGILYA APQLRSIRGQPGPNHEEDADSYENM 16 CD5-FcRγ-CD40 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAA SGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRA NRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDE SPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDI YIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDG VYTGLSTRNQETYETLKHEKPPQKKVAKKPTNKAPHPKQE PQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQ ERQ 24 CD5-FcRγ-MDA5 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAA SGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRA NRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDE SPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDI YIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDG VYTGLSTRNQETYETLKHEKPPQGSGSMSNGYSTDENFRY LISCFRARVKMYIQVEPVLDYLTFLPAEVKEQIQRTVATSG NMQAVELLLSTLEKGVWHLGWTREFVEALRRTGSPLAAR YMNPELTDLPSPSFENAHDEYLQLLNLLQPTLVDKLLVRDV LDKCMEEELLTIEDRNRIAAAENNGNESGVRELLKRIVQKE NWFSAFLNVLRQTGNNELVQELTGSDCSESNAEIEN 25 CD5-FcRγ-TNFR1 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAA SGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRA NRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDE SPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDI YIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDG VYTGLSTRNQETYETLKHEKPPQGSGSQRWKSKLYSIVCG KSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTF TSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIP NPLQKWEDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFV RRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRRE ATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR 26 CD5-FcRγ-TNFR2 MWLQSLLLLGTVACSISEIQLVQSGGGLVKPGGSVRISCAA SGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYAD SFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS LSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRA NRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDE SPWTFGGGTKLEIKSGGGGSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDI YIWAPLAGTCGVLLLSLVITLYCRLKIQVRKAAITSYEKSDG VYTGLSTRNQETYETLKHEKPPQGSGSPLCLQREAKVPHLP ADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRN QPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVN VCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEEC AFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS

TABLE B Linker sequences SEQ ID Sequence 10 SSGGGGSGGGGSGGGGS 11 SGGGGSG 12 SGGG 13 GSGS IV. Noncoding Exogenous Sequence for Delivery and Incorporation into the Genome of a Cell

A noncoding sequence may be delivered into the cell and designed to be incorporated in the genome of the cell. The noncoding sequence as used herein, is a sequence that does not result in a translated protein product, but may have regulatory elements, such as transcribed products, such as inhibitory RNA. In some embodiments, such a sequence may be a miRNA sequence. In some embodiments, the sequence may be a sequence for siRNA generation. In some embodiments, the sequence may comprise an intronic sequence, or a binding site created, such that one or more DNA binding proteins can dock on the site and influence the nature and behavior of the adjoining regions. In some embodiments, the sequence may be a transcription factor binding site. In some embodiments, the sequence may comprise an enhancer binding site. In some embodiments, the sequence may comprise a binding site for topoisomerase, gyrase, reverse transcriptase, polymerase, poly A binding protein, guanylyl cyclase, ligase, restriction enzymes, DNA methylase, HDAC enzymes, and many others. In some embodiments, the noncoding sequence may be directed to manipulating heterochromatin. A noncoding insert sequence, as it may also be referred to here, may be a few nucleotides to 5 kB in length.

V. Plasmid Design and Recombinant Nucleic Acid Design Comprising an Insert Sequence

The nucleic acid construct comprising one or more sequences encoding one or more proteins or polypeptides is incorporated in a plasmid for transcription and generating an mRNA. mRNA can be transcribed in an in vitro system using synthetic system of cell extracts. Alternatively, mRNA can be generated in a cell and harvested. The cell can be a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the transcription occurs in a synthetic system. Provided herein are exemplary plasmid design.

In some embodiments, of the various aspects of the disclosure, a plasmid is designed for expression of the mRNA molecule comprising a heterologous sequence of interest that encodes a protein or a polypeptide. The plasmid comprises, inter alia: the sequences for genomic integration elements for integration of the heterologous sequence of interest that encodes a protein or a polypeptide; the sequence comprising the transgene or fragment thereof, operably linked to its separate promoter and regulatory elements that are required for its expression in the host following integration in the host genome, (such as, the subject who is administered the mRNA); one or more regulatory elements for transcription and generation of the mRNA including a promoter for expression of the mRNA, e.g. in a bacterial cell or cell extract, and 3′ stabilizing elements; sequences for one or more detection marker and/or selection markers.

As is known to one of skill in the art, a plasmid backbone can be an available vector, such as an in-house or commercially developed vector, that can be improved in various ways for best expression of the transcribed sequences, for example, (but not limited to), by introducing one or more desirable restriction digestion sites in the MCS (multiple cloning site), introducing a desired promoter for overall mRNA transcription, such as the T7 promoter, exchanging an existing sequence within the plasmid vector for one or more desired sequences, or introducing one or more desired segments, such as a selection marker sequence.

The plasmid comprises transcription regulatory elements, such as a promoter at the 5′ region, and a 3′-stabilizing element. In some embodiments, the promoter is chosen for enhanced mRNA transcription in the desired cell, such as an E coli bacterial cell. In some embodiments, the promoter for transcription of the plasmid is selected from a T7 promoter, a Sp6 promoter, pL (lambda) promoter, T3 promoter, trp promoter, araBad promoter, lac promoter or a Ptac promoter. In some embodiments, the promoter is a T7 promoter. T7 or Sp6 promoters are constitutive promoters and are useful for high level transcription or in vitro transcription. In some embodiments, the 3′ stabilizing element is a sequence from BGH 3′ element, WPRE 3′ element, SV40 element, hGH element and other elements. The 3′ element comprises the necessary poly A and transcription termination sequences.

Exemplary selection markers include antibiotic selection marker and/or expression detection marker. Antibiotic selection markers include but are not limited to ampicillin resistance gene sequence (beta lactamase gene or fragment thereof) conferring resistance to ampicillin, for example G418 selection marker, tetracycline resistance gene sequence conferring resistance to tetracycline, kanamycin resistance gene sequence conferring resistance to kanamycin, erythromycin resistance gene sequence conferring resistance to erythromycin, chloramphenicol resistance gene sequence conferring resistance to chloramphenicol, neomycin resistant gene sequence conferring resistance to neomycin, and others. Exemplary expression detection marker include FLAG, HA, GFP and others.

In some embodiments, the and other tags that can be fused to one or more coding sequences to function as a surrogate for the expression of the desired protein or peptide to which it is fused.

In some embodiments, the plasmid is less than 20 kb in length. In some embodiments, the plasmid is less than 19 kb in length. In some embodiments, the plasmid is less than 20 kb in length. In some embodiments, the plasmid is less than 18 kb in length. In some embodiments, the plasmid is less than 20 kb in length. In some embodiments, the plasmid is less than 17 kb in length. In some embodiments, the plasmid is less than 20 kb in length. In some embodiments, the plasmid is less than 16 kb in length. In some embodiments, the plasmid is less than 15 kb in length. In some embodiments, the plasmid is less than 14 kb in length. In some embodiments, the plasmid is less than 13 kb in length. In some embodiments, the plasmid is less than 12 kb in length. In some embodiments, the plasmid is about 15 kb, about 14 kb, about 13 kb, about 12 kb or about 10 kb in length.

In some embodiments, the codon is optimized for maximized transcription suitable for the transcription system.

VI. Features Related to the Expression of the Transgene In Vivo Transcription Regulatory Elements in the Recombinant Nucleic Acid Construct (Transgene)

In some embodiments, the recombinant nucleic comprises one or more regulatory elements within the noncoding regions that can be manipulated for desired expression profiles of the encoded proteins. In some embodiments, the noncoding region may comprise suitable enhancer. In some embodiments, the enhancer comprises a binding region for a regulator protein or peptide may be added to the cell or the system comprising the cell, for commencement of expression of the protein encoded under the influence of the enhancer. Conversely, a regulatory element may comprise a protein binding domain that remains bound with the cognate protein and continue to inhibit transcription and/or translation of recombinant protein until an extracellular signal is provided for the protein to decouple from the bound position to allow commencement of the protein synthesis. Examples include but are not limited to Tetracycline-inducible (Tet-Inducible or Tet-on) and Tetracycline repressible (Tet-off) systems known to one of skill in the art.

Construct comprising metabolic switch: In some embodiments, the 5′ and 3′ untranslated regions flanking the coding regions of the construct may be manipulated for regulation of expression of the recombinant protein encoded by the nucleic acid constructs described above. For instance, the 3′UTR may comprise one or more elements that are inserted for stabilizing the mRNA. In some embodiments, AU-Rich Elements (ARE) sequences are inserted in the 3′ UTR that result in binding of RNA binding proteins that stabilize or destabilize the mRNA, allowing control of the mRNA half-life.

In some embodiments, the 3′UTR may comprise a conserved region for RNA binding proteins (e.g. GAPDH) binding to mature mRNA strand preventing translation. In some embodiments, glycolysis results in the uncoupling of the RNA binding proteins (e.g. GAPDH) allowing for mRNA strand translation. The principle of the metabolic switch is to trigger expression of target genes when a cell enters a certain metabolic state. In resting cells, for example, GAPDH is an RNA binding protein (RBP). It binds to ARE sequences in the 3′UTR, preventing translation of mRNA. When the cell enters glycolysis, GAPDH is required to convert glucose into ATP, coming off the mRNA allowing for translation of the protein to occur. In some embodiments, the environment in which the cell comprising the recombinant nucleic acid is present, provides the metabolic switch to the gene expression. For example, hypoxic condition can trigger the metabolic switch inducing the disengaging of GAPDH from the mRNA. The expression of the mRNA therefore can be induced only when the macrophage leaves the circulation and enters into a tumor environment, which is hypoxic. This allows for systemic administration of the nucleic acid or a cell comprising the nucleic acid, but ensures a local expression, specifically targeting the tumor environment.

In some embodiments, the nucleic acid construct can be a split construct, for example, allowing a portion of the construct to be expressed under the control of a constitutive expression system whereas another portion of the nucleic acid is expressed under control of a metabolic switch, as described above. In some embodiments, the nucleic acid may be under bicistronic control. In some embodiments, the bicistronic vector comprises a first coding sequence under a first regulatory control, comprising the coding sequence of a target recognition moiety which may be under constitutive control; and a second coding sequence encoding an inflammatory gene expression which may be under the metabolic switch. In some embodiments, the bicistronic vector may be unidirectional. In some embodiments, the bicistronic vector may be bidirectional.

In some embodiments, the ARE sequences comprise protein binding motifs for binding ARE sequence that bind to ADK, ALDH18A1, ALDH6A1, ALDOA, ASS1, CCBL2, CS, DUT, ENO1, FASN, FDPS, GOT2, HADHB, HK2, HSD17B10, MDH2, NME1, NQ01, PKM2, PPP1CC, SUCLG1, TP11, GAPDH, or LDH.

Pharmaceutical Compositions and Immunotherapy

In one aspect provided herein is a pharmaceutical composition comprising (i) the nucleic acid encoding the transgene is incorporated in a transpositioning or retrotranspositioning system comprising the transgene, the 5′- and 3′-flanking transposition or retrotranspositioning elements, the expression regulation elements, such as promoters, introns; and a nucleic acid encoding the transposase or retrotransposase, (ii) a nucleic acid delivery vehicle and a pharmaceutically acceptable salt or excipient.

In some embodiments, the pharmaceutical composition comprises cells comprising the nucleic acid encoding the transgene that is stably integrated in the genome of the cell and a pharmaceutically acceptable excipient. Nucleic acid constructs can be delivered with cationic lipids (Goddard, et al, Gene Therapy, 4:1231-1236, 1997; Gorman, et al, Gene Therapy 4:983-992, 1997; Chadwick, et al, Gene Therapy 4:937-942, 1997; Gokhale, et al, Gene Therapy 4:1289-1299, 1997; Gao, and Huang, Gene Therapy 2:710-722, 1995), using viral vectors (Monahan, et al, Gene Therapy 4:40-49, 1997; Onodera, et al, Blood 91:30-36, 1998), by uptake of “naked DNA”, and the like. Techniques well known in the art for the transformation of cells (see discussion above) can be used for the ex vivo administration of nucleic acid constructs. The exact formulation, route of administration and dosage can be chosen empirically. (See e.g. Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 pl).

In some embodiments, the nucleic acid comprising the transgene and the transposable elements is introduced or incorporated in the cell by known methods of nucleic acid transfer inside a cell, such as using lipofectamine, or calcium phosphate, or via physical means such as electroporation or nucleofection. In some embodiments, the nucleic acid is encapsulated in liposomes or lipid nanoparticles. LNPs are 100-300 nm in diameter provide efficient means of mRNA delivery to various cell types, including macrophages. In some embodiments, the nucleic acid is transferred by other nanoparticles. In some embodiments, the vector for expression of the CFP is of a viral origin, namely a lentiviral vector or an adenoviral vector. In some embodiments, the nucleic acid encoding the recombinant nucleic acid is encoded by a lentiviral vector. In some embodiments, the lentiviral vector is prepared in-house and manufactured in large scale for the purpose. In some embodiments, commercially available lentiviral vectors are utilized, as is known to one of skill in the art.

In some embodiments, the viral vector is an Adeno-Associated Virus (AAV) vector.

The methods find use in a variety of applications in which it is desired to introduce an exogenous nucleic acid into a target cell and are particularly of interest where it is desired to express a protein encoded by an expression cassette in a target cell, where the target cell or cells are part of a multicellular organism. The transposase system may be administered to the organism or host in a manner such that the targeting construct is able to enter the target cell(s), e.g., via an in vivo or ex vivo protocol. Such cells or organs are typically returned to a living body.

In some embodiments, the transgene encoding a fusion protein related to immune function is stably integrated in a living cell of a subject ex vivo, following which the cell comprising the transgene is returned to the subject. Of exemplary importance, the CFP transgene (phagocytic receptor fusion protein) is intended for expression in an immune cell, such as a myeloid cell, a phagocytic cell, a macrophage, a monocyte or a cell of dendritic cell lineage is contacted ex vivo with the recombinant nucleic acids for stable transfer of the transgene and re-introduced in the same subject for combating a disease of the subject. The diseases contemplated comprises infectious diseases, cancer and autoimmune diseases. The nucleic acid encoding the PSR subunit comprising fusion protein (CFP) described herein is used to generate engineered phagocytic cells for treating cancer.

Cancers include, but are not limited to T cell lymphoma, cutaneous lymphoma, B cell cancer (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), the heavy chain diseases (such as, for example, alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers can be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, or undifferentiated. In some embodiments, the present disclosure is used in the treatment, diagnosis, and/or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

In general, cellular immunotherapy comprises providing the patient a medicament comprising live cells, which should be HLA matched for compatibility with the subject, and such that the cells do not lead to graft versus Host Disease, GVHD. A subject arriving at the clinic for personalized medicine and immunotherapy as described above, is routinely HLA typed for determining the HLA antigens expressed by the subject.

Therapeutic Advantages of mRNA Driven Delivery

In one embodiment, provided herein is a method of introducing a nucleic acid sequence into a cell for sustained gene expression in the cell without adverse effects. In some embodiments, the cell is within a living system, e.g., a host organism such as a human. The nucleic acid sequence is an mRNA.

In particular, delivery via retrotransposon poses to be a highly lucrative mode. mRNA driven delivery simplifies gene delivery. While other technologies require expensive and sophisticated design and manufacturing, and a solution for delivery of the nucleic acid into the cell, and gene editing technologies to assist in integration, retrotransposon mediated delivery itself encodes for the editing machinery, encodes for new genes to be delivered. In addition, a single mRNA may be sufficient for gene delivery and editing.

In one embodiment, mRNA delivery is advantageous in that it can ensure introduction of a nucleic acid cargo without size restraint.

Table 9 summarizes some of the advantages over the other existing methods of nucleic acid deliveries.

TABLE 9 Advantages of retrotransposon mediated gene delivery Retrotransposon Lentiviral delivery AAV-delivery delivery Payload ~4 kb ~4 kb >10 kb Toxicity Insertional Unresolved Unknown, mutagenesis liver & pending clinical CNS toxicity development Manufacturing Complex, Complex, Inexpensive, expensive expensive rapid

Retrotransposons are advantageous for applications across multiple modalities. Gene manipulation using this method is easily attained both in vivo and ex vivo. In one embodiment, the application of retrotransposon may be in vivo, a piece of genetic material encoded in an mRNA can be directly introduced into a patient by systemic or local introduction. In contrast, cells can be taken out from a subject, and manipulated ex vivo and then introduced either to the same subject (autologous) or to another human (heterologous).

In one embodiment, retrotransposons and the related methods described herein may be instrumental in gene therapy. With the advantage of capacity to introduce large payloads, large sections of DNA carrying a gene encoding an entire protein may be introduced in one shot without requiring multiple introductions and multiple editing events. In one embodiment, for example, a gene that encodes a defective protein may be excised, the correct gene may be introduced in the correct site in one integration event using a retrotransposon mediated delivery. In one example, CRISPR editing may be used to excise a gene from precise locus and retrotransposition may be used to replace the correct genes. In some embodiments, a preferred retrotransposon integration site may be introduced at the excision site.

In one embodiment, retrotransposons and the related methods described herein may be instrumental in gene editing.

In one embodiment, retrotransposons and the related methods described herein may be instrumental in transcriptional regulation.

In one embodiment, retrotransposons and the related methods described herein may be instrumental in genome engineering.

In one embodiment, retrotransposons and the related methods described herein may be instrumental in developing cell therapy, for example chimeric antigen receptor (CAR)T cells, in NK cell therapy or in myeloid cell therapy. In one embodiment, retrotransposons and the related methods described herein may be instrumental in delivery of genes into neurons, which are difficult to access by existing technologies.

In one aspect, provided herein is a method for targeted replacement of a genomic nucleic acid sequence of a cell, the method comprising: (A) introducing to the cell a polynucleotide sequence encoding a first protein complex comprising a targeted excision machinery for excising from the genome of the cell a nucleic acid sequence comprising one or more mutations; and (B) a recombinant mRNA encoding a second protein complex, wherein the recombinant mRNA comprises: (i) a nucleic acid sequence comprising the excised nucleic acid sequence in (A) that does not contain the one or more mutations, and (ii) a sequence encoding an L1 retrotransposon ORF2 protein under the influence of an independent promoter.

In one embodiment, the first protein complex may be an endonuclease complex independent of the second protein complex. In one embodiment, the first protein complex comprises a CRISPR-CAS system that uses sequence guided genomic DNA excision. In one embodiment, the methods described herein couples a CRISPR CAS system or any other gene editing system with a Lil transposon machinery (e.g., the second protein complex) that delivers a replacement gene with a payload capacity of greater than 4 kb, or 5 kb, or 6 kb, or 7 kb, or 8 kb or 9 kb or 10 kb. This coupling can be utilized in precisely excising a large fragment (a mutated gene causing a disease) from the genomic locus and integrating a large fragment of a gene or an entire gene that encodes a correct, non-mutated sequence.

A large number of genetic diseases may require delivery of gene delivery of large payloads, often exceeding the functional capacity of existing methods. Contemplated herein are methods and compositions disclosed herein that can be instrumental in further designing therapy for such diseases using retrotransposons. An exemplary list of genetic diseases include but are not limited to the ones listed in Table 10.

TABLE 10 List of potential gene therapy applications Disease Gene CDS Expression Prevalence Stargardt ABCA4 6.8 kb Rod and Cone 1:8000   PRs Usher 1B MY07A 6.7 kb RPE and PRs 3.2:100,000  LCA10 CEP290 7.4 kb PR (pan 1:50,000  retinal) USH1D, CDH23 10.1 kb PR 3:100,000 DFNB12 RP EYS 9.4 kb PR ECM 1:50,000  USH2A USH2a 15.6 kb Rod and Cone 4:100,000 PRs USH2C GPR98 18.0 kb Mainly PRs 1:100,000 Alstrom ALMS1 12.5 kb Rod and Cone  1:1,000,000 syndrome PRs Glycogen GDE 4.6 kb Muscle, Liver 1:8000   storage disease III Non-syndromic OTOF 6.0 kb Ear 14:100,000  deafness Hemophilia A F8 7.1 kb Liver 1:10,000  Leber CEP290 7.5 kb Retina 5:100,000 congenital aumaurosis

Provided herein is a method for targeted replacement of a genomic nucleic acid sequence in a cell. In one embodiment, the method comprises: (A) excising from the genome of the cell a nucleic acid sequence comprising one or more mutations and (B) introducing into the cell a recombinant mRNA encoding: (i) a nucleic acid sequence comprising a wild type sequence relative to the sequence excised in (A) that does not contain the one or more mutation, (ii) a sequence encoding an L1 retrotransposon ORF2 protein under the influence of an independent promoter. In one embodiment, Step (A) further comprises introducing a short sequence comprising at least a plurality of adenylate residues at the excision site. In one embodiment, the In one embodiment, the nucleic acid sequence comprising a wild type sequence is operably linked with the ORF2 encoding sequence in a way such that the ORF2 reverse transcriptase integrates the sequence comprising the wild type non-mutated sequence into the genome.

In one embodiment, the cell is a lymphocyte.

In one embodiment, the cell is an epithelial cell. In some embodiments the cell is a retinal pigmented epithelial cell (RPE).

In one embodiment, the cell is a neuron.

In one embodiment, the cell is a myeloid cell.

In one embodiment, the cell is a stem cell.

In one embodiment, the cell is a cancer cell.

In one embodiment, the gene is selected from a group consisting of ABCA4, MY07A, CEP290, CDH23, EYS, USH2a, GPR98, ALMS1, GDE, OTOF and F8.

In one embodiment, the mRNA comprises a sequence for an inducible promoter.

In one embodiment, the expression of the nucleic acid sequence comprising a non-mutated sequence is detectable at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days post infection.

In one embodiment, the method comprises introducing into the cell a recombinant mRNA in vivo.

In one embodiment, the method comprises introducing into the cell a recombinant mRNA ex vivo.

Provided herein is a method of treating a genetic disease in a subject in need thereof, comprising: introducing into the subject a composition comprising a polycistronic mRNA encoding a gene or fragment thereof, operably linked to a sequence encoding an L1 retrotransposon; wherein the gene or the fragment thereof is at least 10.1 kb in length.

In one embodiment, the method comprises directly introducing the mRNA systemically.

In one embodiment, the method comprises directly introducing the mRNA locally.

In one embodiment, the genetic disease is a retinal disease. For example, the disease is macular dystrophy. In one embodiment, the disease is stargardt disease, also known as juvenile macular degeneration, or fundus flavimaculatus. The disease causes progressive degeneration and damage of the macula. The condition has a genetic basis due to mutation in the ATP-binding cassette (ABC) transporter gene, (ABCA4) gene, and arises from the deposition of lipofuscin-like substance in the retinal pigmented epithelium (RPE) with secondary photoreceptor cell death. In some embodiments, the method comprises direct delivery of the mRNA to the retina.

In one embodiment, the method comprises treating a nonsyndromic autosomal recessive deafness (DFNB12) and deafness associated with retinitis pigmentosa and vestibular dysfunction (USH1D). In one embodiment, provided herein is a method of treating non-syndromic deafness (DFNB12) or Usher syndrome (USH1D), the method comprises introducing an mRNA comprising a copy of CDH23 or a fragment thereof operably linked to a sequence encoding an L1 retrotransposon.

EXAMPLES

It should be appreciated that the invention should not be construed to be limited to the examples which are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.

Example 1. Exemplary Retrotransposon Designs Constructs

Provided here are exemplary strategies of designing retrotransposon constructs for incorporating into the genome of a cell and expressing an exemplary transgene. FIG. 1B and FIG. 1C illustrates various strategic designs for integrating an mRNA encoding transgene into the genome of a cell. GFP shown here in a box is an exemplary transgene. The mRNA encoding the transgene (e.g., GFP) can be co-expressed with a nucleic acid sequence encoding an ORF2p protein, in either sense or antisense orientation; the respective coding sequences may be in a monocistronic or bicistronic construct shown under exemplary Cis-strategies (FIG. 1B and FIG. 1C). CMV/T7 are promoters.

On the other hand, the same could be directed to be expressed in a trans manner. The trans-strategy can include a sequence encoding an ORF2p protein or both ORF1p and ORF2p proteins from a bicistronic sequence and an mRNA encoding a GFP in a sense or antisense direction in the 3′UTR of any gene. The transgene is flanked by a retrotransposing sequence comprising transposase binding sequences, an A-box and B-box, and a poly A tail. FIG. 2A illustrates three exemplary designs for expressing an exemplary transgene GFP by stably incorporating the sequence encoding GFP using the constructs. The first construct comprises a sequence encoding GFP, flanked by L1 5′-UTR; and a poly A sequence at the 3′ UTR, in absence of any transposase binding elements. The second and the third constructs comprise a sequence encoding GFP, a 3′UTR an A Box and a B-box, and a poly A sequence at the 3′ UTR. The third construct comprises an additional sequence encoding ORF2p. Expected GFP expression levels at 72 hours are shown on the right side. FIG. 2B illustrates three exemplary designs for expressing an exemplary transgene GFP in an mRNA that either encodes RFP or ORF2p by stably incorporating the sequence encoding GFP using the constructs. The first construct comprises a sequence encoding RFP, and a poly A sequence at the 3′ UTR, in absence of any L1 elements. The second and the third constructs comprise a 3′UTR comprising an A Box and a B-box, and a poly A sequence at the 3′ UTR. The second construct comprises a sequence encoding RFP and the third construct comprises a sequence encoding ORF2p. Expected RFP and GFP expression levels at 72 hours are shown on the right side.

Example 2. Exemplary circRNA Designs Constructs

In this example, modular designs for circRNA are demonstrated, which incorporate a stretch of about 50 nucleotide long RNA having naturally occurring tertiary structures in order to prepare a circRNA. Use of the tertiary-structure forming RNA makes the circRNA formation process independent of sequence mediated hybridization for circularization. These RNA motifs having tertiary structures can be incorporated in the desired RNA having an exon and an intron in place of the 5′ and 3′ homology arms, thereby forming the terminal RNA scaffolds for circularization.

TectoRNA: RNA-RNA binding interfaces are constructed by combining pairs of GNRA loop/loop-receptor interaction motifs, yielding high affinity, high specificity tertiary structures. (FIG. 3B). Pairs of GNRA loop/loop-receptor interaction motifs are fused using the four-way junction from the hairpin ribozyme to create divalent, self-assembling scaffolding units (‘tectoRNA’) which help form a closed cooperatively assembling ring-shaped complexes. Using two orthogonal loop/loop-receptor interaction motifs, RNA monomers are designed that are capable of directional assembly in either the parallel (‘up-up’) or anti-parallel (‘up-down’) assembly modes. In anti-parallel assembly of interacting molecules, each incorporated monomer switches the directionality of the growing chain and thus compensates for its intrinsic bending, producing long, relatively straight multi-unit chains. For selecting a tectoRNA scaffolds having minimum occurrences of alternative secondary structures, sequences are checked by submitting them to the RNA folding program Mfold (bioinfo.mathspi.edu/˜zukerm/rna/mfold) which predicts the thermodynamically favored secondary structure of a given RNA sequence. A thermodynamically favored structure is selected for scaffolding that has minimum alternative secondary structures (typically but not exclusively, no other secondary structure is closer than 15% in energy to the lowest energy structure). RNA molecule is prepared by conventional methods, such as in vitro run-off transcription using T7 RNA polymerase. FIG. 3B shows a RL-GAAA loop structure. In order to profile tectoRNA heterodimers a fluorescence-based chip-flow piece testing method is utilized. In this method, a library of potential variants of the structured RNA (chip piece) is synthesized as DNA templates and amplified to include sequencing adapters and regions for RNAP initiation. Each DNA variant is transcribed in situ into RNA, enabling display of sequence-identified clusters of RNA on the surface of the sequencing chip. The fluorescently-labeled tectoRNA binding partner, the “flow piece”, is introduced to the sequencing chip flow cell at increasing concentrations, allowing quantification of bound fluorescence to each cluster of RNA after equilibration. These fluorescence values are used to derive the affinity of the flow piece to each chip piece variant (FIG. 3C), in terms of the dissociation constant (K_(d)) and binding free energy, (ΔG=RT log(K_(d)))

The selected terminal RNA scaffold segments comprising the tertiary structures are incorporated using T7 transcription or ligated at the 5′ and 3′ ends of the desired RNA to be circularized; or are incorporated in the desired RNA by any known molecular biology techniques.

Example 3: Exemplary Retrotransposon Designs with Enhanced Specificity

In this example, designs for a nucleic acid construct for L1-mediated retrotransposon for enhanced target specificity is demonstrated. An mRNA is designed comprising ORF2 encoding sequence and a sequence encoding a gene of interest, to incorporate the gene of interest into the genome of a cell using ORF2. In one exemplary design, the construct comprises an ORF2 that is further modified.

As shown in FIG. 4A, ORF2 protein initiates retrotransposition by binding to its own poly A sequence. However, because poly A is abundantly present in mRNAs, a non-specific binding and integration becomes a possibility. To increase the specificity, a recombinant ORF2 is designed comprising an mRNA-binding domain of a heterologous protein, and the cognate mRNA sequence for the heterologous mRNA-binding domain is inserted near the poly A sequence in the 3′-UTR and the ORF2 poly A binding site.

A chimeric ORF2 is thereby generated as shown in (FIG. 4B), in which a high affinity RNA-binding domain of a heterologous protein encoding sequence is incorporated or fused to the ORF2 sequence and cognate RNA sequences corresponding to the high affinity RNA-binding protein is incorporated in the 3 ′UTR region of the mRNA, proximal to the poly A region. In this example the heterologous high affinity RNA-binding domain is derived from MCP coat protein MS2 (shown as M in the figure), is incorporated within the ORF2 sequence and the cognate sequence, the MS2 hairpin, is included in the 3′ UTR sequence of the mRNA (FIG. 4B). The MS2 binds to the cognate sequence, increasing the specificity of the chimeric ORF2 to its own mRNA for reverse transcribing and incorporating the respective sequence associated with the ORF2 mRNA in the mammalian cell genome (FIG. 4B).

In other exemplary designs, attempts to increase specificity of integration of the transgene by the ORF2 within the genome of a target cell is undertaken. In one exemplary design, Mega TAL encoding sequence fused to an ORF2 as shown in FIG. 4C (upper panel). Along with that, the ORF2 is mutated to remove its ability to recognize and bind to RNA sequence that has less specificity. The fused protein is directed to the TAL binding sequence incorporated within the 3′UTR and perform endonuclease function. The Mega TAL DNA binding sequence is targeted by the fusion protein. Likewise, other chimera (FIG. 4C (middle panel)) and fusion protein with a specific DNA binding domain FIG. 4C (lower panel) are designed.

Example 4. Exemplary Plasmid Design and Developments for LINE-1 Mediated Retrotransposition of an Exogenous Nucleic Acid Sequence

In this example plasmid vectors are generated for delivery and incorporation of a recombinant LINE-1 construct comprising an ORF2 transposon element operably linked to a transgene transposable into a mammalian cell, and regulatory elements for mRNA transcription and stabilization. The mRNA can be transcribed in a bacterial host cell, which can be further processed and/or purified for introduction into a mammalian cell in vitro or administration in an organism, such as a mammal, a rodent, sheep, pig or a human.

Any suitable vector backbone is used for incorporating the recombinant nucleic acid sequence as insert and transcribing in a bacterial system for mRNA generation; or in vitro transcription system may be utilized to generate an mRNA comprising the recombinant nucleic acid sequence. Several features are added to the plasmid. Upon successful scalable mRNA production, and purification, the mRNA may be introduced in a mammalian cell of interest, such as a myeloid cell.

Plasmids traditionally used in the field of study for retrotransposition lack designer genes, gene blocks, and Gibson assembly methods were used regularly to insert different features. A new vector that takes features from the old vectors but has flexibility to insert new features can be beneficial both for the study and optimization of LINE-1 elements as a gene delivery system. Below is an outline of base features and additional features that can increase retrotransposition frequency, both using the plasmid alone or the mRNA transcribed from the plasmid. In an exemplary plasmid design shown graphically in FIG. 5(I), which contains the natural LINE-1 sequence with the original 5′UTR, 3′UTR and interORF sequence with no restriction sites to swap out any of these features. New optimized plasmid:

-   -   Removed Dox inducible promoter, replaced with CMV or EIF1a or         EF1a promoter     -   Added a T7 site to make mRNA     -   Codon optimized ORF1 and ORF2     -   Added a WPRE element to stabilize mRNA     -   Added FLAG tag to ORF2 to help with protein detection     -   Decreased size from 18 kb to 14 kb     -   Added blunt restriction sites (dotted lines with blunt arrows)         at each feature to facilitate insertions     -   Includes a G418 selection marker

The plasmid is shown in FIG. 5 (II).

With Gibson a reverse split GFP is inserted for plasmid reporter gene as shown in FIG. 5 (III). A complete reverse GFP for the mRNA reporter is inserted as in FIG. 5 (IV).

Using the plasmid construct in FIG. 5 (V) as parent, a nuclear localization sequence (NLS) is inserted at the N terminus of ORF2 to help with nuclear import (FIG. 5 (VI)). An IRES or another termination/promoter sequence is inserted to increase expression of ORF2 (FIG. 5 (VII)). To facilitate stronger interactions between ORF2 and the mRNA, MS2 hairpins are inserted in the 3′UTR and a MS2 coat protein sequence in the N terminus of the ORF2 protein (FIG. 5 (VIII)). A corresponding exemplary ORF2 with enhanced specificity and its mechanism of action is disclosed in the preceding example and in FIG. 4B. To facilitate stronger interactions of the mRNA with the translating ribosome and to stall translation so that nascent ORF2 will more likely bind the mRNA, an Alu element is inserted in the 3′UTR of the mRNA (FIG. 5 (IX)). To potentially use a more active ORF2 protein, the ORF2's RT domain is replaced with the Group II intron's reverse transcriptase domain (FIG. 5 (X)). Additionally, the minke whale genome has the highest number and percentage of active LINE elements (˜5,000 with 60% active compared to humans that have 480 with 3.6% active). The two sequences are 67% identical and the whale sequence has the active endonuclease and reverse-transcriptase residues. The respective minke whale domains can be used to replace native ORF2 endonuclease and/or RT domains or design a chimera domain.

Example 5. mRNA Design Synthetic mRNA Generation

mRNA can be strategically designed for synthetic production by oligosynthesis and or ligation of oligonucleotides. Additionally, such designs are useful for in vitro transcription (IVT) mediated mRNA generation. The mRNA strategy can include the same variants as the plasmid strategy discussed in the previous example. The main differences are that the reporter GFP sequence does not include an intron (FIG. 6A) and that the constructs can be delivered without the ORF1 coding region (FIG. 6B).

Example 6. Structural Features for Increased mRNA Half-Life

In this example, structural features are introduced in the mRNA comprising the retrotransposition elements and/or the transgene for increasing the mRNA half-life. The goal is to increase the duration of protein expression from the mRNA in primary monocytes from three days to at least 5 days with an ultimate goal of 10 days.

As shown in FIG. 7B (left), the mRNA comprising a sequence encoding the transgene when introduced into a CD14+ myeloid cell (monocyte), is translated and expresses a chimeric receptor (an ATAK construct) capable of binding to an antigen on a cancer cell.

A number of mRNA designs are generated by synthesizing various gene blocks comprising singly, or combinations of one or more of: (i) a G-quadruplex, (ii) a viral pseudoknot structure in the 5′ UTR; and/or (iii) one or (iv) more xrRNA loop structures in the 3′ UTR (v) a triplex RNA structure as shown in FIG. 7A; and cloned into the transcription vector at the respective UTRs adjoining the coding sequence of the transgene. These constructs are individually prepared by an off-site vendor and tested in-house for determining stability of the mRNA, as measured by the expression of the chimeric receptor (An exemplary receptor and its function is depicted graphically in FIG. 7B (left). The process flow chart is shown on FIG. 7B (right). In short, constructs are cloned into plasmids, with encoded or modified poly A tails. The mRNA was transcribed and purified. Meanwhile, frozen monocytes are thawed and harvested. Harvested cells were electroporated with the purified mRNA (5-10 ug), and cultured for 1, 2, 3, 5 days. Cells positive for the chimeric receptor (binder positive cells), are detected by means of their ability to bind to a target cell or a substrate coated with the target antigen. The expected results are shown in FIG. 7C. Bulk or purified mRNA expressing one or more of the structural features outlined in (i)-(v) (data denoted by solid squares) or a combination thereof outperforms the commercially available counterparts that do not contain any of the features outlined in (i)-(v) (data denoted by triangles).

Example 7. LINE-1 Retrotransposon Plasmid Mediated Delivery of GFP Gene

In this test run, genomic integration of a GFP cargo and expression the GFP protein using a LINE-1 retrotransposon system was verified. The LINE-1-GFP construct (LINE-1 plasmid GFP) is exemplified in FIG. 8A: A plasmid construct having a LINE-1 sequence encoding ORF1p (ORF1), a sequence encoding ORF2p (ORF2), and a CMV promoter driven split GFP gene situated in the 3′UTR of the LINE-1 in reverse orientation with respect to the ORFs. The split GFP is designed to have an intronic sequence inserted in between a splice donor and acceptor sites, which ensures that the GFP is expressed only after integration and splicing mediated removal of the noncoding sequence in the middle of the coding sequence. In this case the cargo is 2.1 kb. HEK293T cells were transfected with the plasmid using Fugene reagent, and plasmid positive cells were selected by puromycin. The mRNA generated from a genome integrated GFP successfully translates and is measured by flow cytometry, as indicated as change in mean fluorescence intensity (MFI) (FIG. 8B) and fraction of cells with GFP fluorescence intensity compared to mock transfected cells (FIG. 8C). Mock transfected cells received the plasmid that lack the GFP sequence.

Example 8. LINE-1 Retrotransposon Plasmid Mediated Delivery of a Chimeric Receptor Gene

This example demonstrates that a recombinant gene can be successfully expressed using the LINE-1 sequence in a cell. HEK 293 cells were transfected with a plasmid having the LINE-1 elements, with a 3 kb cargo sequence encoding recombinant receptor protein CD5-intron-fcr-PI3K (ATAK) that is interrupted by an intron sequence in the CD5 binding domain. The cargo is a chimeric receptor that has a CD5 binding extracellular domain, a FCRγ transmembrane domain, and an intracellular domain having a PI3-kinase recruitment domain. The schematic representation of the retrotransposon plasmid is shown in FIG. 9A. As in the design of the experiment above, the ATAK receptor cannot express unless it is integrated in the genome and the intron is spliced off. Following transfection in HEK293T cells, the receptor expression is detected using labeled CD5 as bait for the CD5 binding extracellular domain. Results shown in FIGS. 9B and 9C show successful integration and expression of the receptor. 36.5% cells were ATAK (CD5 binder) positive (FIG. 9C).

In a further modification, a LINE-1 construct (LINE-1plasmid-cd5_fcr-pi3k_t2a_GFPintron) with a longer 3.7 kb cargo sequence encoding a non-interrupted recombinant receptor protein CD5-intron-fcr-PI3K and an interrupted GFP sequence with a T2A sequence between receptor and the GFP sequences (FIG. 10A). Normalized against mock-transfected cells, there was a greater than 10-fold increase of the ATAK receptor and GFP double-positive cells was noted (FIG. 10B). Exemplary fluorescence identification of GFP and fluorescent tagged CD5 binding and gating quantitation for experimental runs are shown in FIG. 10C and FIG. 10D.

Example 9. mRNA Encoding LINE-1 Retrotransposon for Delivery of a Cargo Gene

In this assay, capability of delivering and expressing a LINE-1 retrotransposable gene sequence as an mRNA was tested. An mRNA encoding an ORF1 (ORF1-FLAG-mRNA), and an mRNA encoding ORF2 and GFP in the antisense direction with a CMV promoter sequence (ORF2-FLAG-GFPai) are designed as shown in FIG. 11A. The cargo size in this assay was 2.4 kb, and GFP is in antisense orientation with respect to ORF2 sequence. The mRNAs were electroporated in 293T cells and the reporter genes expression was demonstrated as shown in FIG. 11B. This experimental set up demonstrated that no ORF1-readthrough is necessary for the expression of the ORF2p, and expression of ORF2p from a different mRNA molecule can allow higher expression of ORF2p and GFP. With these results, a successful delivery of the LINE-1 and cargo in the form of mRNA was achieved.

In order to determine whether the relative levels of ORF1 and ORF2 mRNA affected GFP expression an experiment was set up to test the varying amounts of ORF1 and ORF2 mRNAs (FIG. 11A). 3× the amount of each and together is tested for increases in GFP+ cells and results are shown in FIG. 12A. Fold increase is relative to 1×ORF2-GFP and 1×ORF1 mRNA. GFP expression was higher when 3×ORF1 was used with 1×ORF2, but not the reverse; whereas having both 3×ORF1 and 3×ORF2 showed the maximum level of GFP expression in the sets compared. The cargo size here is 2.4 kb. FIG. 12B shows fluorescent microscopy image of GFP+ cells following retrotransposon mRNA electroporation.

A complete LINE-1 mRNA encoding both ORF1 and ORF2 and GFP transgene in antisense orientation in a single mRNA molecule (LINE 1-GFP mRNA construct) was tested for delivery and genomic integration in a cell. mRNA contains the bicistronic ORF1 and ORF2 sequence with a CMV-GFP sequence in the 3′UTR going from 3′-5′ (FIG. 13A). In this study the cargo size is 2.4 kb. As shown in FIG. 13B, upon retrotransposition of the delivered ORF2-cmv-GFP antisense (LINE-1 mRNA), third bar from left, cells expressed higher GFP compared to ORF1 and ORF2 being on separate mRNA molecules (graph bar 1, 2). Inclusion of ORF1 in a separate mRNA in addition to LINE-1 complete mRNA increased GFP expression over LINE-1 alone. Inclusion of ORF2+ GFP expectantly showed higher GFP which could be the contribution of the additional ORF2 with the GFP cargo encoding mRNA.

To test whether subsequent electroporation increases retrotransposition efficiency, cells were electroporation every 48 hours. GFP positive cells were assessed using flow after culturing for 24-72 hrs. The fluorescence data were normalized to the values in the set with a single electroporation event. As shown in FIGS. 14A and 14B, multiple electroporation led to an upward trend in the expression of the transposed gene, but the changes were modest.

Example 10. Modifications to the ORF2 Protein Sequence to Enhance Retrotransposition by mRNA

Modification of the LINE-1 sequence to enhance retrotransposition via mRNA delivery were tested using GFP reporter as readout. The experiment was performed as follows. All modifications were in the context of the bicistronic ORF1 and ORF2 sequence. (i) ORF2-NLS fusion was created by inserting C-terminal NLS sequence to the ORF2 sequence. (ii) Human ORF2 was replaced with Minke whale ORF2; (Ivancevic et al., 2016). (iii) Incorporation of an Alu element in the 3′UTR: Using a minimal sequence of the Alu element (AJL-H33Δ; Ahl et al., 2015) in the 3′UTR of the LINE-1. (iv) MS2-hairpin in the 3′UTR+ORF2-MCP fusion: MS2 hairpins in the 3′UTR of the LINE-1 sequence and a MS2 hairpin binding protein (MCP) fused to the ORF2 sequence (FIG. 15A). The mock construct had the wild-type human ORF2 sequence.

Quantification of the fold increase in the fraction of GFP positive cells relative to mock construct electroporated cells are shown in FIG. 15B.

Example 11. Retrotransposition in an Immune Cell

In this experiment, the inter-ORF region is further manipulated to determine if any of the changes improve GFP expression after transfection of the HEK cells. Taking LINE-1plasmid GFP, the inter-ORF region is manipulated as follows: (a) In one construct the inter-ORF region is replaced with an IRES from CVB3; (b) In another construct, the inter-ORF region is replaced with an IRES from EV71; (c) In three separate constructs, an E2A or P2A or T2A self-cleavage sequence is intercalated in the inter-ORF region. Result are as shown in FIG. 16. Compared to the LINE-1 plasmid GFP (LINE-1 wild type plasmid) led to only modest changes in the GFP readout, especially with T2A sequence insertion. Insertion of EV71 IRES sequence improved GFP expression, while CVB3 IRES did not show any improvement.

Example 12. Retrotransposition in an Immune Cell

To test retrotransposition in immune cells, LINE-1 plasmid and mRNA were tested with the CMV-GFP antisense reporter cargo by electroporating into Jurkat cells, which is a T cell lymphoma line (FIG. 17A-FIG. 17B). Mock set were electroporated with a plasmid with no GFP sequence. GFP expression in the transfected cells was assessed, representative data at 4 days post electroporation is shown in FIG. 17B. Fold increase is reported relative to mock transfected cells. Both plasmid and mRNA delivery modes resulted in successful GFP expression.

Next, THP-1 cells (a myeloid, monocytic cell line) were electroporated with a plasmid having LINE-1 sequences and a 3.7 kb cargo encoding a chimeric HER-2 binding receptor, and a split GFP (LINE-1 plasmid Her2-Cd3z-T2A-GFPintron) (FIG. 18A). The cargo is a chimeric receptor that comprises a HER2 binding extracellular domain, a CD3z transmembrane domain, and split GFP reporter. The plasmid was successfully integrated into the genome and showed prolonged expression, as demonstrated in FIG. 18B. Representative expression at day 6 post transfection is shown in the figure. From these studies, it was demonstrated that LINE-1 mediated gene delivery can result in successful stable genomic integration in various cell types, including epithelial cell types (HEK-293T cells); T cells (e.g., Jurkat cells); and cells of myeloid lineage (e.g., THP-1 cells) and results in prolonged expression. Moreover, unlike CRISPR dependent technologies such as Prime editing, retrotransposition can result in integration of large genetic cargo, and, these can be delivered as a single nucleic acid construct.

Example 13. External Methods for Further Enhancing Efficiency of LINE-1 Mediated Retrotransposition of the Cargo Sequences

In this section, methods for further enhancing the efficiency of retrotransposition of cargo sequences into the genome of cells are detailed.

Cell cycle synchronization by selection of cells in a population that are in a certain stage of cell cycle or G1 arrest by a suitable agent can lead to higher nucleic acid uptake efficiency, e.g., plasmid vector transfection efficiency or electroporation efficiency. In this assay, cells are pre-sorted and each group is separately electroporated to ensure uniform electroporation. The efficiencies of electroporation are compared between these groups and a cell cycle stage that results in highest efficiency as determined by the expression of the GFP test plasmid or mRNA is selected (FIG. 19).

In another variation of this experiment, cells are synchronized with or without sorting by treating the cells, with a cell cycle arrest reagent for a few hours prior to electroporation. An exemplary list of cell cycle arrest reagents is provided in Table 1. The list is non-exhaustive, and is inclusive of reagents that can be proapoptotic, and hence careful selection suitable for the purpose and dose and time of incubation is optimized for use in the particular context.

TABLE 1 Exemplary non-exhaustive list of small molecule reagents that are used for inhibiting cell cycle Agent Cell cycle Mechanism 5-[(4- Arrests cell cycle at Inhibits c-Myc-Max Ethylphenyl)methyl- G0-G1 dimerization ene]-2-thioxo-4- thiazolidinone Itraconazole Inhibits cell cycle at SMO antagonist G1 ABT 751 (Tocris Blocks cell cycle at Inhibits microtubule Bioscience, cat #4138) G2M proliferation Artesunate Arrests cell cycle at Suppresses ROS- G2M induced NLRP3 AZD 5438 Blocks cell cycle at Inhibits Cdk G2M, M, S and G1 phases Baicalein Arrests cell cycle at Inhibits G1 and G2 phases lipoxygenases CPI 203 (alternative Arrests cell cycle at BET bromodomain name: TEN 101) G1 phase inhibitor Diadzein Arrests cell cycle at Estrogen receptor G1 agonist DIM Blocks cell cycle at Induces EGFR G2M activation Epothilone B Arrests cell cycle at Inhibits tubulin G2M proliferation Indirubin-3′-oxime Antiproliferative Inhibits GSK3b MPC 6827 Cell cycle arrest Inhibits microtubule hydrochloride proliferation Pladienolide Inhibits G1 and Decreases mRNA G2/M splicing Plumbagin Induces G2/M arrest Inhibits TOR signaling and others Temsirolimus Induces G1/S mTOR inhibitor Toceranib Cell cycle arrest Inhibits PDGFR and VEGFR WYE 687 Induces G1 arrest mTOR inhibitor dihydrochloride YC1 Induces G1 arrest Guanylyl cyclase activator

For certain ex vivo usages, retrotransposition is enhanced by inducing DNA double stranded breaks (DSB) in a cell that expresses a retrotransposition machinery as described in any of the examples above by controlled irradiation, which create opportunities for the homologous recombination and priming for the reverse transcriptase (FIG. 20). In another example, cells transfected with LINE-1 plasmid GFP construct and subjected to an irradiation pulse. GFP expression is monitored. The intensity and time of irradiation is optimized for obtaining the maximum benefit, as indicated by higher GFP expression.

In another example, cells transfected with LINE-1 plasmid GFP were divided into experimental sets that are treated as follows (i) irradiation in order to induce DSB (as described above); (ii) treat cells in this set with a small molecule, such as SCR7, that blocks DNA ligase and therefore inhibits the DNA damage repair machinery. Preventing protective repair mechanism from inhibiting the progress of the retrotransposition is expected to enhance GFP expression: (iii) irradiate the cells then treat the cells with SCR7, combination of the two is expected to show a more robust effect. GFP expression is monitored over a period of 6 days, and the set that shows maximum GFP fluorescence over the longest period indicates a condition that is adopted in further studies.

Example 14. Enhancing Efficiency of LINE-1 Mediated Retrotransposition of the Cargo Sequences by Further Modification of the Construct

I. Enhancing non-coding regions of the construct to offer stability and higher expression. In this example a LINE-1 plasmid-GFP is further modified to test for increased GFP expression as follows: (a) In one construct, the 5′UTR is replaced with an UTR of a complement gene; (b) In another construct, the 3′UTR is replaced with the UTR sequence of B-globin gene for increased stability; (c) In another construct the inter-ORF region is replaced with an IRES from CVB3; (d) In another construct, the inter-ORF region is replaced with an IRES from EV71 (e) In three separate constructs, an E2A or P2A or T2A self-cleavage sequence is intercalated in the inter-ORF region as shown in a diagrammatic representation in FIG. 21. In addition to the above, various combinations of (a)-(e) and additional combinations listed in Table 2 are tested using the same set-up as above. GFP expressions are monitored after transfection of the constructs in parallel test sets into HEK293T cells to see if any of these constructs increased GFP expression compared to the LINE-1 plasmid GFP alone. The combinations that show improvement are adopted.

TABLE 2 Exemplary combinations of 5′ and 3′ UTR and inter-ORF insertion elements for inclusion in the LINE-1 construct for increase in retrotransposition efficiency. 5′-UTR sequences selected from sequences 3′-UTR sequences Inter-ORF sequences Complement 5′UTR WPRE T2A, E2A, P2A Covid-19 5′ leader B-globin 3′UTR CVB3 IRES sequence CYBA 5′UTR RSV RSE EV71 IRES CYP2E1 5′ UTR AREs EMCV IRES RNA zipcodes for the PV IRES ER mtRNR1-AES CSFV IRES HRV2 IRES AAA (tri alanine fusion or any fusion- linker sequence)

II. Enhancing localization and retention of the ORFs in the nucleus. In this example, LINE-1 plasmid-GFP is further modified to test for increased GFP expression as follows: (a) the ORF2 encoding sequence is fused with a nuclear localization sequence (NLS) (graphically represented in FIG. 15A second construct from top). (b) the ORF1 encoding sequence is fused with a nuclear localization sequence (NLS), graphically represented in FIG. 22; and (c) An Alu binding sequence is inserted 3′ of the sequence encoding ORF2 reverse transcriptase (graphically represented in FIG. 15A, fourth construct from the top; (d) Both (a) and (c) together (not shown); (e) Both (b) and (c) together, the NLS sequence is fused to the ORF1 N-terminus, and an Alu binding sequence is inserted 3′ of the sequence encoding ORF2 reverse transcriptase (FIG. 22) and (f) Integrating a SINE-derived nuclear RNA LOcalizatIoN (SIRLOIN) sequence in LINE-1 3′ UTR. HEK-293T cells were transfected with constructs (a)-(f) and the LINE-plasmid GFP construct in parallel. GFP expression is monitored after transfection into HEK293T cells. The set that shows maximum GFP fluorescence over the longest period is adopted.

III. Modifying construct to increase LINE-1 protein-RNA complex binding to the ribosome. In this example, an additional sequence is inserted in the 3′UTR of the LINE-1 construct to increase association of the LINE-1 protein RNA construct to the ribosomes, the sequence is an Alu element, or a ribosome binding aptamer (FIG. 23).

For enhancing LINE-1 protein-RNA complex binding to the ribosome, insertion of the following elements in the 3′ UTR of the mRNA is done and tested similar to the experiments above. Insertion of Alu elements is described above. In separate constructs, Alu element truncations, Ribosome binding aptamers (109.2-3) and Ribosome expansion segments (ES9S) binding sequence are inserted and each tested for increase in GFP expression.

IV. Enhancing binding of ORF2 to its own mRNA for retrotransposition. In this example, a sequence containing MS2 binding loop structure is introduced into the 3′UTR of the LINE-1, and a sequence encoding MS2 RNA binding domain is fused to the RNA binding domain of the ORF2p-RT (graphically represented in FIGS. 4A and 4B, and FIG. 24, construct SEQ ID NO: 15). The fused protein will specifically attach to the MS2-binding structural motif in the 3′ UTR, and therefore any non-specific binding and retrotransposition is minimized (FIG. 24). GFP expression is monitored after transfection into HEK293T cells. Following a similar design, the ORF is fused with the protein binding sequences shown in left column of Table 3 below, combined with a cognate sequence inserted in the 3′UTR region of the ORF2 shown in the corresponding right column in the same row.

TABLE 3 Exemplary list of elements to enhance translation efficiency and stability of the LINE-1 proteins and increased expression of LINE-1 proteins. Elements to be fused with the 3′ UTR sequence recognizable by LINE-1 ORF2 the element PP7 coat protein PP7 Streptavidin S1m aptamer Tobramycin Tobramycin aptamer

V. Modifying the endonuclease function of the retrotransposon. In this example, the constructs are modified to test increase in GFP expression as follows. In a first experimental set, the LINE-1 plasmid GFP is cut at the 3′ end of the endonuclease coding sequence of ORF2, and a sequence encoding the DNA binding domain (DBD) of a heterologous zinc finger protein (ZFP) is inserted. In another experimental set, the endonuclease domain is fused with a CRISPR nuclease. A variety of nucleases can be tested by modifying the LINE-1 plasmid GFP ORF by creating a fusion protein using DNA binding domains and cleavage domain as shown in a non-exhaustive list in Table 4, In addition, two ORF-2 domains are encoded in one set to facilitate dimerization. The construct that has higher GFP expression than the ORF2 endonuclease can be further selected. The plasmid designs are graphically represented in FIG. 25. GFP expression is monitored after transfection of the plasmids into HEK293T cells, and the set that yielded best.

TABLE 4 Exemplary non-exhaustive list of additional DNA cleavage domains/enzymes that can be fused to or inserted in place of LINE-1 endonuclease. Gene/ Enzyme Description Fold Class II endonuclease from Flavobacterium okeanokoites, recognition and cleavage sequence are separated by a few nucleotides; recognizes DNA sequence 5-GGATG-3′ Restriction e.g., HindII, EcoR1, BamH1 enzymes, LAGLIDADG Intron encoded homing proteins found in various genera family including bacteria nuclease A GIY-YIG This domain is found in the amino terminal region of excinuclease abc subunit c (uvrC), bacteriophage T4, endonuclease segA, segB, seg C, seg D, and seg E and group I introns of fungi and phage. His-Cys box Homing endonucleases containing two clusters of conserved histidine and cysteine residues over a 100 amino acid region. H-N-H Widely present nuclease in phage DNA. Crucial component of the terminase packaging reaction of E. coli phage HK97. PD-(D/E)xK Phosphodiesterases, present in a large number of proteins, e.g., DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII. Vsr-like/ C-terminal nuclease domain that displays recognizable EDxHD homology to bacterial Very short repair (Vsr) endonucleases

VI. Modifying the reverse transcriptase function of the retrotransposon. In this example, the reverse transcriptase domain of ORF2 is modified for increasing its efficiency. In one experimental set, the sequence encoding the human ORF2 in LINE-1plasmid GFP is excised and replaced with a sequence encoding MMLV or TGIRTII. In another experimental set, the ORF2 reverse transcriptase domain is fused with a DNA binding domain of a heterologous protein. The reverse transcriptase domains and/or the DNA binding domains can be selected from a non-exhaustive list provided in Table 5A-Table 5B. The constructs are graphically exemplified in FIG. 26. GFP expression is monitored after transfection into HEK293T cells.

TABLE 5A Selected non-exhaustive list of reverse transcriptase for replacing the LINE-1 RT for higher efficiency Reverse Transcriptase Description M-MLV-RT Murine leukemia virus TGIRT-II Thermostable group II intron reverse transcriptase with high fidelity and processivity AMV-RT Avian Myeloblastosis Virus reverse transcriptase Group II intron Derived from Eubacterium rectale maturase RT HIV-RT Efficient RT derived from HIV TERT Catalyzes the RNA-dependent extension of 3′- chromosomal termini with the 6-nucleotide telomeric repeat unit, 5′-TTAGGG-3′.

TABLE 5B Selected non-exhaustive list of DNA-binding domains for fusing to a RT for higher efficiency DNA binding domains (DBD) Zinc finger domains Leucine zipper (bZip) Helix-turn-helix domain HMG-box R2 retroelement DBD Sso7d Protein A (ssDNA) OB-fold (ssDNA)

VII. Replacing human LINE-1 with LINE-1 from other organisms. In this example, the sequence encoding human LINE-1 is replaced by a LINE-1 from a different organism. In one example, the human LINE-1 construct is compared with a construct where the human LINE-1 is replaced by a minke whale LINE-1 sequence (FIG. 27). Using the same experimental framework, a number of ORFs are tested. An exemplary non-exhaustive list is provided in Table 6 below. A further comprehensive list is available in Ivancevic A. et al., Genome Biol Evol 8(11):3301-3322.

TABLE 6 Exemplary LINE-1 elements from organism for use in replacement of the human LINE-1 No of total LINE-1/No active/ Species Name percent active Balaenoptera acutorostrata scammoni 8,012/5,006/62.4% Rhinopithecus roxellana 11,115/2,954/26.5% Mus musculus 18,280/4,143/22.66% Aedes aegypti 519/184/35.4% Zea mays 744/165/22.17% Brassica napus 1,929/565/29.2% Brassica rapa 543/228/41.9% Danio rerio 590/268/45.4%

In another set, human LINE-1 is retained as in the GFP plasmid, but an inhibitor of human LINE-1 silencer is utilized to prevent recognition by endogenous proteins like HUSH complex TASOR protein. In this case, the TASOR inhibitor is an inhibitory RNA, such as a miRNA.

VIII. LINE-1 fusion proteins for target specificity. In this example, the LINE-1 plasmid GFP ORF2 is fused with a domain of a MegaTAL nuclease, a CRISPR-CAS nuclease, a TALEN, R2 retroelement binding zinc finger binding domain, or a DNA binding domain that can bind to repetitive elements such as Rep78 AAV. FIG. 28 exemplifies the deigns. Table 7 provides a list of the different elements that can be fused to increase sequence specific retrotransposition.

TABLE 7 Exemplary proteins with DNA binding domains to be fused to ORF2 for increasing retrotransposition specificity Elements Transcription Factors MegaTAL nucleases TALENs Zinc finger binding domains from other retroelements Safe harbor binding proteins Cfp1

Each plasmid is transfected into HEK293 cells and GFP expression is monitored.

The modifications described in this section under (I)-(VIII) are designed to test for increase in retrotransposition efficiency, using GFP as readout. Following this, a number of useful modifications from (I)-(VIII) are incorporated into a single retrotransposition construct, tested with GFP as insert for the outcome, and the GFP sequence is replaced by the desired insert sequence.

Example 15. Delivering a Large Payload for Prolonged Expression Using Retrotransposon Technology

Provided here are exemplary demonstrations of retrotransposon constructs are versatile for incorporating nucleic acid payloads into the genome of a cell and expressing an exemplary transgene. Retrotransposon constructs were designed as elaborated elsewhere in the disclosure.

Briefly, in one set of validation experiments, GFP encoding payloads were constructed as follows: an antisense promoter sequence under doxycycline inducible control followed by antisense GFP gene split with an intron in the sense direction was placed downstream of the LINE-1 ORFs (FIG. 29). Splicing donor (SD) and splicing acceptor (SA) sequences are recognized and spliced out only when the mRNA is produced from the promoter in the top strand, therefore only the GFP gene integrated into genome from spliced mRNA generates fluorescent signal. As shown in the representative flow cytometry data in FIG. 2, the GFP expression was measured 35 days post doxycycline induction of the ORF expression using flow cytometry (green histogram) compared to a negative control plasmid (grey histogram). In this case, the cargo size was 2.4 kb.

The cargo GFP gene in the previous construct was replaced with intron interrupted CD5-FcR-PI3K CAR-M sequence (Morrissey et al., 2018). The CD5 binder expression was measured by flow cytometry using a Alexa647-conjugated CD5 protein such that retrotransposed cells are CD5-AF647 positive (red histogram) compared with a plasmid transfected negative control cell population (grey histogram) (FIG. 30). Successful expression of the 3.0 kb construct was demonstrated as shown in the figure.

The cargo gene length was extended by adding the intron-interrupted GFP gene after the T2A sequence downstream of the CD5-FcR-PI3K CAR-M sequence (FIG. 31). The CD5 binder expression was measured by flow cytometry using a Alexa647-conjugated CD5 protein. The CD5 binder positive cells shown by red histogram, in comparison with a negative control (grey histogram). The GFP expression is measured using flow cytometry (green histogram) compared to a negative control plasmid transfected cells (grey histogram). The flow cytometry signal in the Q2 showed that 10.8% cells express both CAR-M and GFP proteins.

As shown in FIG. 32, the payload size limit has not been reached with retrotransposon delivery and integration (Retro-T delivery) with a 3.9 kb payload. The delivery mechanism described here was successful for expression of the first generation CART construct and GFP (separated by T2A site). In this example, different constructs were tested for retrotransposition efficiency of the insert sequence. FIG. 33A shows gene delivery as mRNA results in successful integration. This data is the first to show that Retro T can be delivered as mRNA. A trans strategy of using separate mRNAs encoding for ORF1 and ORF2 with antisense promoter and GFP cargo (ORF2-GFPai) in the 3′ UTR for gene delivery was explored, as exemplified graphically in FIG. 33B (top panel). FIGS. 33B-33D demonstrate experimental results from multiple representative assays. Separate mRNAs that expression the LINE-1 proteins could reconstitute the RNA-protein complex required for retrotransposition. The cis strategy uses a single bicistronic LINE-1 mRNA with the antisense promoter and GFP gene cargo in the 3′UTR. Constructs comprising variable amounts and proportions of ORF2 and ORF1 were compared as shown in FIG. 33B and FIG. 33C with GFP encoding sequence as payload. FIG. 33D shows that introducing a single mRNA yields higher number of integrations per cell. Sorting of 293T GFP cells to enrich for retrotransposed cells for biochemical and integration assays. Cells are the same as in FIG. 33B and show GFP expression 4 days post-sort in bottom panels. The graph shows qPCR assay for genomic DNA integration from different LINE-1 plasmid transfected, LINE-1 mRNA (retro-mRNA), and ORF1 and ORF2-GFP mRNA electroporated cells. Two qPCR primer-probe sets were used, one for the housekeeping gene RPS30 and the other for the GFP gene. Plasmid-transfected cells use a plasmid that does not contain and SV40 maintenance sequence. Integration per cell is calculated from determining copy numbers per samples through interpolation of a standard curve of plasmid and genomic DNA and normalizing for the two copies of RPS30 per 293T cell. Error bar denote standard deviation of three technical replicate measurements.

Example 16. Delivery to Diverse Cell Types

As shown in FIGS. 34-38, the mRNA constructs comprising a gene of interest, e.g. encoding a CAR protein, or for example, a GFP protein can be efficiently expressed in diverse cell types, such as epithelial cells (e.g., HEK 293 cells), monocytic cells lines (e.g., THP-1 cells), lymphoblastic cell lines (e.g., K562 cells), and primary lymphocytes (T cells). Activated primary T cells were also successfully transfected with mRNA with genomic integration and expression of GFP (FIG. 36). Primary T cells were isolated and expanded using IL7/IL15; and a 1^(st) Gen CAR construct was delivered on day 2 post activation. Cells sorted and frozen. GFP expression was detectable after a freeze-thaw cycle (FIG. 37A-B). This indicates the versatile nature of mRNA mediated delivery and L1-transposon mediated integration. FIG. 38 shows a representative assay of GFP mRNA integration and expression in 293T cells, K562 cells, THP-1 cells and Primary T cells.

EXEMPLARY SEQUENCES

Following are exemplary sequences of the constructs used in the examples. These sequences are for reference exemplary purposes and sequence variations and optimizations that are conceivable by one of skill in the art without undue experimentation are contemplated and encompassed by the disclosure. Where mRNA sequences are referred in the sequence title, the construct recites nucleotides of a DNA template and one of skill in the art can easily derive the corresponding mRNA sequence.

TABLE 8 Plasmid and mRNA construct sequences ORF1-FLAG-mRNA (Codon Optimized human ORF1 coding sequence-FLAG) (SEQ ID NO: 35): 1 TAATACGACT CACTATAGGG AGAAAGACGC CACCATGGGC AAGAAGCAAA ATCGCAAGAC 61 GGGGAATTCC AAGACACAAT CCGCTAGCCC ACCACCTAAA GAGCGTTCTA GCTCCCCTGC 121 TACTGAGCAG TCCTGGATGG AAAACGACTT CGATGAACTC CGGGAAGAGG GATTTAGGCG 181 ATCCAACTAT TCAGAACTCC GCGAAGATAT CCAGACAAAG GGGAAGGAAG TCGAGAATTT 241 CGAGAAGAAC CTCGAGGAGT GCATCACCCG TATCACAAAC ACTGAGAAAT GTCTCAAAGA 301 ACTCATGGAA CTTAAGACAA AAGCCAGGGA GCTTCGAGAG GAGTGTCGGA GTCTGAGATC 361 CAGGTGTGAC CAGCTCGAGG AGCGCGTGAG CGCGATGGAA GACGAGATGA ACGAGATGAA 421 AAGAGAGGGC AAATTCAGGG AGAAGCGCAT TAAGAGGAAC GAACAGAGTC TGCAGGAGAT 481 TTGGGATTAC GTCAAGAGGC CTAACCTGCG GTTGATCGGC GTCCCCGAGA GCGACGTAGA 541 AAACGGGACT AAACTGGAGA ATACACTTCA AGACATCATT CAAGAAAATT TTCCAAACCT 601 GGCTCGGCAA GCTAATGTGC AAATCCAAGA GATCCAACGC ACACCCCAGC GGTATAGCTC 661 TCGGCGTGCC ACCCCTAGGC ATATTATCGT GCGCTTTACT AAGGTGGAGA TGAAAGAGAA 721 GATGCTGCGA GCCGCTCGGG AAAAGGGAAG GGTGACTTTG AAGGGCAAAC CTATTCGGCT 781 GACGGTTGAC CTTAGCGCCG AGACACTCCA GGCACGCCGG GAATGGGGCC CCATCTTTAA 841 TATCCTGAAG GAGAAGAACT TCCAGCCACG AATCTCTTAC CCTGCAAAGT TGAGTTTTAT 901 CTCCGAGGGT GAGATTAAGT ATTTCATCGA TAAACAGATG CTGCGAGACT TCGTGACAAC 961 TCGCCCAGCT CTCAAGGAAC TGCTCAAAGA GGCTCTTAAT ATGGAGCGCA ATAATAGATA 1021 TCAACCCTTG CAGAACCACG CAAAGATGGA TTATAAGGAT GACGATGATA AATGA (SEQ ID NO: 35) ORF2-FLAG-GFPai mRNA (Codon Optimized human ORF2 coding sequence) (SEQ ID NO: 36) 1 TAATACGACT CACTATAGGG AGAAAGACGC CACCATGACA GGTTCAAATA GTCACATTAC 61 GATTCTCACT CTGAATATAA ATGGGCTGAA TTCTGCAATT AAACGGCACA GGCTTGCTTC 121 CTGGATAAAG TCTCAAGACC CCTCAGTGTG CTGTATTCAG GAAACGCATC TCACGTGCAG 181 GGACACCCAT CGGCTGAAAA TAAAAGGCTG GCGGAAGATC TACCAAGCCA ATGGAAAACA 241 AAAGAAGGCT GGGGTGGCGA TACTTGTAAG CGATAAAACA GACTTTAAAC CAACTAAGAT 301 CAAACGGGAC AAAGAGGGCC ATTACATCAT GGTAAAGGGT AGTATTCAAC AAGAGGAGCT 361 GACTATCCTG AATATTTATG CACCTAATAC TGGAGCCCCC AGATTCATAA AGCAAGTGTT 421 GAGTGACCTT CAACGCGACC TCGACTCCCA CACTCTGATC ATGGGAGACT TTAACACCCC 481 GCTGTCCACT CTCGACAGAT CTACTAGACA GAAAGTCAAC AAGGATACAC AGGAACTGAA 541 CAGTGCTCTC CACCAAGCGG ACCTTATCGA CATCTACAGA ACACTCCACC CCAAAAGCAC 601 AGAATATACC TTCTTTTCAG CCCCTCACCA CACCTATTCC AAAATTGACC ACATTGTGGG 661 GAGTAAAGCC CTTCTCTCCA AATGTAAACG GACCGAAATT ATCACTAACT ATCTCTCCGA 721 CCACAGTGCA ATAAAACTTG AATTGCGAAT TAAGAATCTC ACTCAAAGTA GATCCACGAC 781 ATGGAAACTG AACAATCTCC TCTTGAATGA CTACTGGGTG CATAACGAAA TGAAGGCTGA 841 AATAAAGATG TTCTTTGAGA CCAACGAAAA CAAAGACACC ACGTACCAGA ATCTCTGGGA 901 CGCTTTCAAA GCAGTGTGTC GAGGAAAATT TATTGCACTG AATGCTTACA AGCGGAAGCA 961 GGAAAGATCC AAAATAGACA CCCTGACTAG CCAACTTAAA GAACTGGAAA AGCAAGAGCA 1021 AACTCATAGC AAAGCTAGCC GTCGCCAAGA AATTACGAAA ATCAGAGCTG AACTGAAGGA 1081 AATTGAGACA CAGAAAACCC TGCAAAAGAT AAATGAAAGC CGCAGCTGGT TCTTTGAACG 1141 CATCAACAAA ATCGATAGGC CACTTGCTCG CCTTATCAAG AAGAAAAGGG AGAAGAATCA 1201 AATCGACACT ATAAAGAATG ATAAAGGCGA TATAACCACC GATCCCACAG AAATTCAAAC 1261 AACCATACGC GAATACTACA AACACCTCTA CGCCAATAAA CTCGAAAATC TCGAGGAAAT 1321 GGATACATTC CTCGACACGT ACACCCTTCC CAGGCTGAAC CAGGAAGAAG TTGAATCACT 1381 GAATCGGCCT ATCACGGGGA GTGAAATAGT AGCTATCATC AATTCACTCC CTACCAAGAA 1441 GTCACCCGGA CCTGATGGAT TCACCGCCGA ATTCTACCAG AGATACATGG AAGAACTGGT 1501 GCCCTTCTTG CTGAAACTTT TCCAAAGTAT TGAGAAAGAG GGAATACTTC CAAACTCATT 1561 TTATGAGGCA TCCATCATTC TGATCCCGAA GCCCGGCAGG GACACGACCA AGAAAGAGAA 1621 TTTTCGACCA ATCTCATTGA TGAACATTGA TGCAAAGATC CTCAATAAAA TACTGGCAAA 1681 TCGGATTCAG CAGCACATAA AGAAGCTGAT CCACCATGAT CAAGTAGGCT TCATCCCCGG 1741 TATGCAAGGT TGGTTCAATA TACGAAAATC AATCAATGTT ATCCAGCATA TAAACCGGGC 1801 CAAAGACAAG AACCACATGA TTATTAGTAT CGATGCTGAG AAAGCCTTTG ACAAAATACA 1861 ACAACCCTTC ATGCTGAAAA CATTGAATAA GCTGGGAATT GATGGCACCT ACTTCAAAAT 1921 CATCAGAGCC ATATATGACA AACCAACAGC AAATATCATT CTGAATGGTC AGAAATTGGA 1981 AGCATTCCCC TTGAAAACCG GCACACGGCA GGGTTGCCCT CTGTCACCAC TCCTCTTCAA 2041 CATCGTGTTG GAAGTTCTTG CCCGCGCAAT CCGGCAGGAA AAGGAAATCA AGGGCATTCA 2101 ACTGGGCAAA GAGGAAGTTA AATTGAGCCT GTTTGCAGAC GACATGATCG TCTATTTGGA 2161 AAACCCCATA GTTAGTGCAC AAAATCTGCT GAAGTTGATC AGTAATTTCT CCAAAGTGAG 2221 TGGGTACAAA ATCAATGTGC AAAAGAGCCA AGCTTTCTTG TACACCAACA ACAGGCAAAC 2281 TGAGTCTCAA ATCATGGGCG AACTCCCCTT CGTGATTGCA TCCAAGCGGA TCAAATACCT 2341 GGGGATTCAA TTGACTCGTG ATGTGAAGGA CCTCTTCAAG GAGAACTACA AACCCCTGCT 2401 CAAGGAAATC AAAGAGGACA CAAACAAATG GAAGAACATT CCATGCTCTT GGGTGGGAAG 2461 GATCAATATC GTCAAAATGG CCATCCTGCC CAAGGTAATT TACAGGTTCA ATGCTATACC 2521 CATCAAGCTC CCCATGACAT TCTTCACAGA ACTTGAAAAG ACGACGCTGA AGTTCATTTG 2581 GAACCAGAAA CGTGCCAGGA TTGCTAAATC TATTCTCTCC CAAAAGAACA AAGCTGGCGG 2641 AATCACACTC CCAGACTTCA AACTTTACTA CAAGGCGACC GTGACGAAAA CGGCTTGGTA 2701 CTGGTACCAA AACAGGGATA TAGATCAATG GAACCGAACG GAGCCCAGCG AAATTATGCC 2761 TCATATATAC AACTATCTGA TCTTTGACAA ACCGGAGAAG AACAAGCAAT GGGGAAAGGA 2821 TAGTCTGTTT AATAAATGGT GCTGGGAAAA CTGGCTCGCA ATCTGTAGGA AGCTGAAACT 2881 GGATCCATTC TTGACGCCTT ATACAAAGAT AAATTCCCGA TGGATTAAAG ATCTCAACGT 2941 GAAACCCAAA ACAATTAAAA CCCTCGAGGA AAACCTGGGT ATTACGATTC AGGACATTGG 3001 GGTGGGAAAG GACTTCATGT CCAAAACCCC AAAAGCGATG GCAACCAAAG ACAAAATCGA 3061 CAAATGGGAT CTCATAAAAC TTAAGTCATT TTGCACAGCT AAAGAAACGA CAATTAGGGT 3121 GAACCGACAA CCGACCACTT GGGAGAAAAT CTTCGCAACA TACAGTTCTG ACAAAGGCCT 3181 GATTTCCAGG ATCTACAATG AATTGAAACA AATTTACAAG AAGAAGACGA ACAACCCTAT 3241 AAAGAAATGG GCCAAGGACA TGAACAGACA CTTCTCTAAG GAAGACATTT ATGCAGCCAA 3301 GAAACACATG AAGAAATGCA GCTCTTCACT GGCAATCAGG GAAATGCAAA TCAAAACAAC 3361 AATGAGATAT CATCTCACAC CCGTCAGAAT GGCCATCATT AAGAAGAGCG GAAACAACCG 3421 GTGCTGGCGT GGTTGCGGAG AAATCGGTAC TCTCCTTCAC TGTTGGTGGG ACTGTAAACT 3481 CGTTCAACCA CTGTGGAAGT CTGTGTGGCG GTTCCTCAGA GATCTGGAAC TCGAAATCCC 3541 ATTTGACCCA GCCATCCCTC TCCTGGGTAT ATACCCGAAT GAGTATAAAT CCTGCTGCTA 3601 TAAAGACACC TGCACAAGGA TGTTTATTGC AGCTCTCTTC ACAATCGCGA AGACGTGGAA 3661 CCAACCCAAA TGTCCGACTA TGATTGACTG GATTAAGAAG ATGTGGCACA TATACACTAT 3721 GGAATACTAT GCTGCGATCA AGAACGATGA GTTCATATCA TTTGTGGGCA CATGGATGAA 3781 ACTCGAAACC ATCATACTCT CTAAATTGAG TCAAGAACAG AAAACTAAAC ACCGTATATT 3841 TTCCCTGATC GGTGGGAATT AGCTACAAAG ACGATGACGA CAAGGACCAT GGAGACGGTG 3901 AGAGACACAA AAAATTCCAA CACACTATTG CAATGAAAAT AAATTTCCTT TATTAGCCAG 3961 AAGTCAGATG CTCAAGGGGC TTCATGATGT CCCCATAATT TTTGGCAGAG GGAAAAAGAT 4021 CTCAGTGGTA TTTGTGAGCC AGGGCATTGG CCTTCTGATA GGCAGCCTGC ACCTGAGGAG 4081 TGCGGCCGCT TTACTTGTAC AGCTCGTCCA TGCCGAGAGT GATCCCGGCG GCGGTCACGA 4141 ACTCCAGCAG GACCATGTGA TCGCGCTTCT CGTTGGGGTC TTTGCTCAGG GCGGACTGGG 4201 TGCTCAGGTA GTGGTTGTCG GGCAGCAGCA CGGGGCCGTC GCCGATGGGG GTGTTCTGCT 4261 GGTAGTGGTC GGCGAGCTGC ACGCTGCCGT CCTCGATGTT GTGGCGGATC TTGAAGTTCA 4321 CCTTGATGCC GTTCTTCTGC TTGTCGGCCA TGATATAGAC GTTGTGGCTG TTGTAGTTGT 4381 ACTCCAGCTT GTGCCCCAGG ATGTTGCCGT CCTCCTTGAA GTCGATGCCC TTCAGCTCGA 4441 TGCGGTTCAC CAGGGTGTCG CCCTCGAACT TCACCTCGGC GCGGGTCTTG TAGTTGCCGT 4501 CGTCCTTGAA GAAGATGGTG CGCTCCTGGA CGTAGCCTTC GGGCATGGCG GACTTGAAGA 4561 AGTCGTGCTG CTTCATGTGG TCGGGGTAGC GGCTGAAGCA CTGCACGCCG TAGGTCAGGG 4621 TGGTCACGAG GGTGGGCCAG GGCACGGGCA GCTTGCCGGT GGTGCAGATG AACTTCAGGG 4681 TCAGCTTGCC GTAGGTGGCA TCGCCCTCGC CCTCGCCGGA CACGCTGAAC TTGTGGCCGT 4741 TTACGTCGCC GTCCAGCTCG ACCAGGATGG GCACCACCCC GGTGAACAGC TCCTCGCCCT 4801 TGCTCACCAT GGTGGCGGGA TCTGACGGTT CACTAAACCA GCTCTGCTTA TATAGACCTC 4861 CCACCGTACA CGCCTACCGC CCATTTGCGT CAATGGGGCG GAGTTGTTAC GACATTTTGG 4921 AAAGTCCCGT TGATTTTGGT GCCAAAACAA ACTCCCATTG ACGTCAATGG GGTGGAGACT 4981 TGGAAATCCC CGTGAGTCAA ACCGCTATCC ACGCCCATTG ATGTACTGCC AAAACCGCAT 5041 CACCATGGTA ATAGCGATGA CTAATACGTA GATGTACTGC CAAGTAGGAA AGTCCCATAA 5101 GGTCATGTAC TGGGCATAAT GCCAGGCGGG CCATTTACCG TCATTGACGT CAATAGGGGG 5161 CGTACTTGGC ATATGATACA CTTGATGTAC TGCCAAGTGG GCAGTTTACC GTAAATACTC 5221 CACCCATTGA CGTCAATGGA AAGTCCCTAT TGGCGTTACT ATGGGAACAT ACGTCATTAT 5281 TGACGTCAAT GGGCGGGGGT CGTTGGGCGG TCAGCCAGGC GGGCCATTTA CCGTAAGTTA 5341 TGTAACGACG TCTCAGCTGA CAATGAGATC ACATGGACAC AGGAAGGGGA ATATCACACT 5401 CTGGGGACTG TGGTGGGGTC GGGGGAGGGG GGAGGGATAG CATTGGGAGA TATACCTAAT 5461 GCTAGATGAC ACATTAGTGG GTGCAGCGCA CCAGCATGGC ACATGTATAC ATATGTAACT 5521 AACCTGCACA ATGTGCACAT GTACCCTAAA ACTTAGAGTA TAATGGATCC GCAGGCCTCT 5581 GCTAGCTTGA CTGACTGAGA TACAGCGTAC CTTCAGCTCA CAGACATGAT AAGATACATT 5641 GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA AATGCTTTAT TTGTGAAATT 5701 TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA ATAAACAAGT T (SEQ ID NO: 36) LINE-1 plasmid GFP (SEQ ID NO: 37) 1 CGGCCGCGGG GGGAGGAGCC AAGATGGCCG AATAGGAACA GCTCCGGTCT ACAGCTCCCA 61 GCGTGAGCGA CGCAGAAGAC GGTGATTTCT GCATTTCCAT CTGAGGTACC GGGTTCATCT 121 CACTAGGGAG TGCCAGACAG TGGGCGCAGG CCAGTGTGTG TGCGCACCGT GCGCGAGCCG 181 AAGCAGGGCG AGGCATTGCC TCACCTGGGA AGCGCAAGGG GTCAGGGAGT TCCCTTTCCG 241 AGTCAAAGAA AGGGGTGACG GACGCACCTG GAAAATCGGG TCACTCCCAC CCGAATATTG 301 CGCTTTTCAG ACCGGCTTAA GAAACGGCGC ACCACGAGAC TATATCCCAC ACCTGGCTCG 361 GAGGGTCCTA CGCCCACGGA ATCTCGCTGA TTGCTAGCAC AGCAGTCTGA GATCAAACTG 421 CAAGGCGGCA ACGAGGCTGG GGGAGGGGCG CCCGCCATTG CCCAGGCTTG CTTAGGTAAA 481 CAAAGCAGCA GGGAAGCTCG AACTGGGTGG AGCCCACCAC AGCTCAAGGA GGCCTGCCTG 541 CCTCTGTAGG CTCCACCTCT GGGGGCAGGG CACAGACAAA CAAAAAGACA GCAGTAACCT 601 CTGCAGACTT AAGTGTCCCT GTCTGACAGC TTTGAAGAGA GCAGTGGTTC TCCCAGCACG 661 CAGCTGGAGA TCTGAGAACG GGCAGACTGC CTCCTCAAGT GGGTCCCTGA CCCCTGACCC 721 CCGAGCAGCC TAACTGGGAG GCACCCCCCA GCAGGGGCAC ACTGACACCT CACACGGCAG 781 GGTATTCCAA CAGACCTGCA GCTGAGGGTC CTGTCTGTTA GAAGGAAAAC TAACAACCAG 841 AAAGGACATC TACACCGAAA ACCCATCTGT ACATCACCAT CATCAAAGAC CAAAAGTAGA 901 TAAAACCACA AAGATGGGGA AAAAACAGAA CAGAAAAACT GGAAACTCTA AAACGCAGAG 961 CGCCTCTCCT CCTCCAAAGG AACGCAGTTC CTCACCAGCA ACAGAACAAA GCTGGATGGA 1021 GAATGATTTT GATGAGCTGA GAGAAGAAGG CTTCAGACGA TCAAATTACT CTGAGCTACG 1081 GGAGGACATT CAAACCAAAG GCAAAGAAGT TGAAAACTTT GAAAAAAATT TAGAAGAATG 1141 TATAACTAGA ATAACCAATA CAGAGAAGTG CTTAAAGGAG CTGATGGAGC TGAAAACCAA 1201 GGCTCGAGAA CTACGTGAAG AATGCAGAAG CCTCAGGAGC CGATGCGATC AACTGGAAGA 1261 AAGGGTATCA GCAATGGAAG ATGAAATGAA TGAAATGAAG CGAGAAGGGA AGTTTAGAGA 1321 AAAAAGAATA AAAAGAAATG AGCAAAGCCT CCAAGAAATA TGGGACTATG TGAAAAGACC 1381 AAATCTACGT CTGATTGGTG TACCTGAAAG TGATGTGGAG AATGGAACCA AGTTGGAAAA 1441 CACTCTGCAG GATATTATCC AGGAGAACTT CCCCAATCTA GCAAGGCAGG CCAACGTTCA 1501 GATTCAGGAA ATACAGAGAA CGCCACAAAG ATACTCCTCG AGAAGAGCAA CTCCAAGACA 1561 CATAATTGTC AGATTCACCA AAGTTGAAAT GAAGGAAAAA ATGTTAAGGG CAGCCAGAGA 1621 GAAAGGTCGG GTTACCCTCA AAGGAAAGCC CATCAGACTA ACAGCGGATC TCTCGGCAGA 1681 AACCCTACAA GCCAGAAGAG AGTGGGGGCC AATATTCAAC ATTCTTAAAG AAAAGAATTT 1741 TCAACCCAGA ATTTCATATC CAGCCAAACT AAGCTTCATA AGTGAAGGAG AAATAAAATA 1801 CTTTATAGAC AAGCAAATGT TGAGAGATTT TGTCACCACC AGGCCTGCCC TAAAAGAGCT 1861 CCTGAAGGAA GCGCTAAACA TGGAAAGGAA CAACCGGTAC CAGCCGCTGC AAAATCATGC 1921 CAAAATGTAA AGACCATCAA GACTAGGAAG AAACTGCATC AACTAATGAG CAAAATCACC 1981 AGCTAACATC ATAATGACAG GATCAACTTC ACACATAACA ATATTAACTT TAAATATAAA 2041 TGGACTAAAT TCTGCAATTA AAAGACACAG ACTGGCAAGT TGGATAAAGA GTCAAGACCC 2101 ATCAGTGTGC TGTATTCAGG AAACCCATCT CACGTGCAGA GACACACATA GGCTCAAAAT 2161 AAAAGGATGG AGGAAGATCT ACCAAGCCAA TGGAAAACAA AAAAAGGCAG GGGTTGCAAT 2221 CCTAGTCTCT GATAAAACAG ACTTTAAACC AACAAAGATC AAAAGAGACA AAGAAGGCCA 2281 TTACATAATG GTAAAGGGAT CAATTCAACA AGAGGAGCTA ACTATCCTAA ATATTTATGC 2341 ACCCAATACA GGAGCACCCA GATTCATAAA GCAAGTCCTC AGTGACCTAC AAAGAGACTT 2401 AGACTCCCAC ACATTAATAA TGGGAGACTT TAACACCCCA CTGTCAACAT TAGACAGATC 2461 AACGAGACAG AAAGTCAACA AGGATACCCA GGAATTGAAC TCAGCTCTGC ACCAAGCAGA 2521 CCTAATAGAC ATCTACAGAA CTCTCCACCC CAAATCAACA GAATATACAT TTTTTTCAGC 2581 ACCACACCAC ACCTATTCCA AAATTGACCA CATAGTTGGA AGTAAAGCTC TCCTCAGCAA 2641 ATGTAAAAGA ACAGAAATTA TAACAAACTA TCTCTCAGAC CACAGTGCAA TCAAACTAGA 2701 ACTCAGGATT AAGAATCTCA CTCAAAGCCG CTCAACTACA TGGAAACTGA ACAACCTGCT 2761 CCTGAATGAC TACTGGGTAC ATAACGAAAT GAAGGCAGAA ATAAAGATGT TCTTTGAAAC 2821 CAACGAGAAC AAAGACACCA CATACCAGAA TCTCTGGGAC GCATTCAAAG CAGTGTGTAG 2881 AGGGAAATTT ATAGCACTAA ATGCCTACAA GAGAAAGCAG GAAAGATCCA AAATTGACAC 2941 CCTAACATCA CAATTAAAAG AACTAGAAAA GCAAGAGCAA ACACATTCAA AAGCTAGCAG 3001 AAGGCAAGAA ATAACTAAAA TCAGAGCAGA ACTGAAGGAA ATAGAGACAC AAAAAACCCT 3061 TCAAAAAATC AATGAATCCA GGAGCTGGTT TTTTGAAAGG ATCAACAAAA TTGATAGACC 3121 GCTAGCAAGA CTAATAAAGA AAAAAAGAGA GAAGAATCAA ATAGACACAA TAAAAAATGA 3181 TAAAGGGGAT ATCACCACCG ATCCCACAGA AATACAAACT ACCATCAGAG AATACTACAA 3241 ACACCTCTAC GCAAATAAAC TAGAAAATCT AGAAGAAATG GATACATTCC TCGACACATA 3301 CACTCTCCCA AGACTAAACC AGGAAGAAGT TGAATCTCTG AATCGACCAA TAACAGGCTC 3361 TGAAATTGTG GCAATAATCA ATAGTTTACC AACCAAAAAG AGTCCAGGAC CAGATGGATT 3421 CACAGCCGAA TTCTACCAGA GGTACAAGGA GGAACTGGTA CCATTCCTTC TGAAACTATT 3481 CCAATCAATA GAAAAAGAGG GAATCCTCCC TAACTCATTT TATGAGGCCA GCATCATTCT 3541 GATACCAAAG CCGGGCAGAG ACACAACCAA AAAAGAGAAT TTTAGACCAA TATCCTTGAT 3601 GAACATTGAT GCAAAAATCC TCAATAAAAT ACTGGCAAAC CGAATCCAGC AGCACATCAA 3661 AAAGCTTATC CACCATGATC AAGTGGGCTT CATCCCTGGG ATGCAAGGCT GGTTCAATAT 3721 ACGCAAATCA ATAAATGTAA TCCAGCATAT AAACAGAGCC AAAGACAAAA ACCACATGAT 3781 TATCTCAATA GATGCAGAAA AAGCCTTTGA CAAAATTCAA CAACCCTTCA TGCTAAAAAC 3841 TCTCAATAAA TTAGGTATTG ATGGGACGTA TTTCAAAATA ATAAGAGCTA TCTATGACAA 3901 ACCCACAGCC AATATCATAC TGAATGGGCA AAAACTGGAA GCATTCCCTT TGAAAACCGG 3961 CACAAGACAG GGATGCCCTC TCTCACCGCT CCTATTCAAC ATAGTGTTGG AAGTTCTGGC 4021 CAGGGCAATC AGGCAGGAGA AGGAAATAAA GGGTATTCAA TTAGGAAAAG AGGAAGTCAA 4081 ATTGTCCCTG TTTGCAGACG ACATGATTGT TTATCTAGAA AACCCCATCG TCTCAGCCCA 4141 AAATCTCCTT AAGCTGATAA GCAACTTCAG CAAAGTCTCA GGATACAAAA TCAATGTACA 4201 AAAATCACAA GCATTCTTAT ACACCAACAA CAGACAAACA GAGAGCCAAA TCATGGGTGA 4261 ACTCCCATTC ACAATTGCTT CAAAGAGAAT AAAATACCTA GGAATCCAAC TTACAAGGGA 4321 TGTGAAGGAC CTCTTCAAGG AGAACTACAA ACCACTGCTC AAGGAAATAA AAGAGGAGAC 4381 AAACAAATGG AAGAACATTC CATGCTCATG GGTAGGAAGA ATCAATATCG TGAAAATGGC 4441 CATACTGCCC AAGGTAATTT ACAGATTCAA TGCCATCCCC ATCAAGCTAC CAATGACTTT 4501 CTTCACAGAA TTGGAAAAAA CTACTTTAAA GTTCATATGG AACCAAAAAA GAGCCCGCAT 4561 TGCCAAGTCA ATCCTAAGCC AAAAGAACAA AGCTGGAGGC ATCACACTAC CTGACTTCAA 4621 ACTATACTAC AAGGCTACAG TAACCAAAAC AGCATGGTAC TGGTACCAAA ACAGAGATAT 4681 AGATCAATGG AACAGAACAG AGCCCTCAGA AATAATGCCG CATATCTACA ACTATCTGAT 4741 CTTTGACAAA CCTGAGAAAA ACAAGCAATG GGGAAAGGAT TCCCTATTTA ATAAATGGTG 4801 CTGGGAAAAC TGGCTAGCCA TATGTAGAAA GCTGAAACTG GATCCCTTCC TTACACCTTA 4861 TACAAAAATC AATTCAAGAT GGATTAAAGA TTTAAACGTT AAACCTAAAA CCATAAAAAC 4921 CCTAGAAGAA AACCTAGGCA TTACCATTCA GGACATAGGC GTGGGCAAGG ACTTCATGTC 4981 CAAAACACCA AAAGCAATGG CAACAAAAGA CAAAATTGAC AAATGGGATC TAATTAAACT 5041 AAAGAGCTTC TGCACAGCAA AAGAAACTAC CATCAGAGTG AACAGGCAAC CTACAACATG 5101 GGAGAAAATT TTTGCAACCT ACTCATCTGA CAAAGGGCTA ATATCCAGAA TCTACAATGA 5161 ACTCAAACAA ATTTACAAGA AAAAAACAAA CAACCCCATC AAAAAGTGGG CGAAGGACAT 5221 GAACAGACAC TTCTCAAAAG AAGACATTTA TGCAGCCAAA AAACACATGA AGAAATGCTC 5281 ATCATCACTG GCCATCAGAG AAATGCAAAT CAAAACCACT ATGAGATATC ATCTCACACC 5341 AGTTAGAATG GCAATCATTA AAAAGTCAGG AAACAACAGG TGCTGGAGAG GATGCGGAGA 5401 AATAGGAACA CTTTTACACT GTTGGTGGGA CTGTAAACTA GTTCAACCAT TGTGGAAGTC 5461 AGTGTGGCGA TTCCTCAGGG ATCTAGAACT AGAAATACCA TTTGACCCAG CCATCCCATT 5521 ACTGGGTATA TACCCAAATG AGTATAAATC ATGCTGCTAT AAAGACACAT GCACACGTAT 5581 GTTTATTGCG GCACTATTCA CAATAGCAAA GACTTGGAAC CAACCCAAAT GTCCAACAAT 5641 GATAGACTGG ATTAAGAAAA TGTGGCACAT ATACACCATG GAATACTATG CAGCCATAAA 5701 AAATGATGAG TTCATATCCT TTGTAGGGAC ATGGATGAAA TTGGAAACCA TCATTCTCAG 5761 TAAACTATCG CAAGAACAAA AAACCAAACA CCGCATATTC TCACTCATAG GTGGGAATTG 5821 AACAATGAGA TCACATGGAC ACAGGAAGGG GAATATCACA CTCTGGGGAC TGTGGTGGGG 5881 TCGGGGGAGG GGGGAGGGAT AGCATTGGGA GATATACCTA ATGCTAGATG ACACATTAGT 5941 GGGTGCAGCG CACCAGCATG GCACATGTAT ACGGATCCGA ATTCTCGACG GATCGATCCG 6001 AACAAACGAC CCAACACCCG TGCGTTTTAT TCTGTCTTTT TATTGCCGAT CCCCTCAGAA 6061 GAACTCGTCA AGAAGGCGAT AGAAGGCGAT GCGCTGCGAA TCGGGAGCGG CGATACCGTA 6121 AAGCACGAGG AAGCGGTCAG CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC 6181 CAACGCTATG TCCTGATAGC GGTCGGCCGC TTTACTTGTA CAGCTCGTCC ATGCCGAGAG 6241 TGATCCCGGC GGCGGTCACG AACTCCAGCA GGACCATGTG ATCGCGCTTC TCGTTGGGGT 6301 CTTTGCTCAG GGCGGACTGG GTGCTCAGGT AGTGGTTGTC GGGCAGCAGC ACGGGGCCGT 6361 CGCCGATGGG GGTGTTCTGC TGGTAGTGGT CGGCCAGGTG AGTCCAGGAG ATGTTTCAGC 6421 ACTGTTGCCT TTAGTCTCGA GGCAACTTAG ACAACTGAGT ATTGATCTGA GCACAGCAGG 6481 GTGTGAGCTG TTTGAAGATA CTGGGGTTGG GGGTGAAGAA ACTGCAGAGG ACTAACTGGG 6541 CTGAGACCCA GTGGCAATGT TTTAGGGCCT AAGGAATGCC TCTGAAAATC TAGATGGACA 6601 ACTTTGACTT TGAGAAAAGA GAGGTGGAAA TGAGGAAAAT GACTTTTCTT TATTAGATTT 6661 CGGTAGAAAG AACTTTCATC TTTCCCCTAT TTTTGTTATT CGTTTTAAAA CATCTATCTG 6721 GAGGCAGGAC AAGTATGGTC ATTAAAAAGA TGCAGGCAGA AGGCATATAT TGGCTCAGTC 6781 AAAGTGGGGA ACTTTGGTGG CCAAACATAC ATTGCTAAGG CTATTCCTAT ATCAGCTGGA 6841 CACATATAAA ATGCTGCTAA TGCTTCATTA CAAACTTATA TCCTTTAATT CCAGATGGGG 6901 GCAAAGTATG TCCAGGGGTG AGGAACAATT GAAACATTTG GGCTGGAGTA GATTTTGAAA 6961 GTCAGCTCTG TGTGTGTGTG TGTGTGTGTG TGTGTGAGAG CGTGTGTTTC TTTTAACGTT 7021 TTCAGCCTAC AGCATACAGG GTTCATGGTG GCAAGAAGAT AACAAGATTT AAATTATGGC 7081 CAGTGACTAG TGCTGCAAGA AGAACAACTA CCTGCATTTA ATGGGAAAGC AAAATCTCAG 7141 GCTTTGAGGG AAGTTAACAT AGGCTTGATT CTGGGTGGAA GCTGGGTGTG TAGTTATCTG 7201 GAGGCCAGGC TGGAGCTCTC AGCTCACTAT GGGTTCATCT TTATTGTCTC CTTTCATCTC 7261 AACAGCTGCA CGCTGCCGTC CTCGATGTTG TGGCGGATCT TGAAGTTCAC CTTGATGCCG 7321 TTCTTCTGCT TGTCGGCCAT GATATAGACG TTGTGGCTGT TGTAGTTGTA CTCCAGCTTG 7381 TGCCCCAGGA TGTTGCCGTC CTCCTTGAAG TCGATGCCCT TCAGCTCGAT GCGGTTCACC 7441 AGGGTGTCGC CCTCGAACTT CACCTCGGCG CGGGTCTTGT AGTTGCCGTC GTCCTTGAAG 7501 AAGATGGTGC GCTCCTGGAC GTAGCCTTCG GGCATGGCGG ACTTGAAGAA GTCGTGCTGC 7561 TTCATGTGGT CGGGGTAGCG GCTGAAGCAC TGCACGCCGT AGGTCAGGGT GGTCACGAGG 7621 GTGGGCCAGG GCACGGGCAG CTTGCCGGTG GTGCAGATGA ACTTCAGGGT CAGCTTGCCG 7681 TAGGTGGCAT CGCCCTCGCC CTCGCCGGAC ACGCTGAACT TGTGGCCGTT TACGTCGCCG 7741 TCCAGCTCGA CCAGGATGGG CACCACCCCG GTGAACAGCT CCTCGCCCTT GCTCACCATG 7801 GTGGCGAATT CGAAGCTTGA GCTCGAGATC TGAGTCCGGT AGCGCTAGCG GATCTGACGG 7861 TTCACTAAAC CAGCTCTGCT TATATAGACC TCCCACCGTA CACGCCTACC GCCCATTTGC 7921 GTCAATGGGG CGGAGTTGTT ACGACATTTT GGAAAGTCCC GTTGATTTTG GTGCCAAAAC 7981 AAACTCCCAT TGACGTCAAT GGGGTGGAGA CTTGGAAATC CCCGTGAGTC AAACCGCTAT 8041 CCACGCCCAT TGATGTACTG CCAAAACCGC ATCACCATGG TAATAGCGAT GACTAATACG 8101 TAGATGTACT GCCAAGTAGG AAAGTCCCAT AAGGTCATGT ACTGGGCATA ATGCCAGGCG 8161 GGCCATTTAC CGTCATTGAC GTCAATAGGG GGCGTACTTG GCATATGATA CACTTGATGT 8221 ACTGCCAAGT GGGCAGTTTA CCGTAAATAC TCCACCCATT GACGTCAATG GAAAGTCCCT 8281 ATTGGCGTTA CTATGGGAAC ATACGTCATT ATTGACGTCA ATGGGCGGGG GTCGTTGGGC 8341 GGTCAGCCAG GCGGGCCATT TACCGTAAGT TATGTAACGC GGAACTCCAT ATATGGGCTA 8401 TGAACTAATG ACCCCGTAAT TGATTACTAT TAGCCCGGGG GATCCAGACA TGATAAGATA 8461 CATTGATGAG TTTGGACAAA CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA 8521 AATTTGTGAT GCTATTGCTT TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA 8581 CAACAATTGC ATTCATTTTA TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG 8641 CAAGTAAAAC CTCTACAAAT GTGGTATGGC TGATTATGAT CCGGCTGCCT CGCGCGTTTC 8701 GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG 8761 TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT 8821 CGGGGCGCAG CCATGAGGTC GATCGACTCT AGAGGATCGA TCCCCGCCCC GGACGAACTA 8881 AACCTGACTA CGACATCTCT GCCCCTTCTT CGCGGGGCAG TGCATGTAAT CCCTTCAGTT 8941 GGTTGGTACA ACTTGCCAAC TGGGCCCTGT TCCACATGTG ACACGGGGGG GGACCAAACA 9001 CAAAGGGGTT CTCTGACTGT AGTTGACATC CTTATAAATG GATGTGCACA TTTGCCAACA 9061 CTGAGTGGCT TTCATCCTGG AGCAGACTTT GCAGTCTGTG GACTGCAACA CAACATTGCC 9121 TTTATGTGTA ACTCTTGGCT GAAGCTCTTA CACCAATGCT GGGGGACATG TACCTCCCAG 9181 GGGCCCAGGA AGACTACGGG AGGCTACACC AACGTCAATC AGAGGGGCCT GTGTAGCTAC 9241 CGATAAGCGG ACCCTCAAGA GGGCATTAGC AATAGTGTTT ATAAGGCCCC CTTGTTAACC 9301 CTAAACGGGT AGCATATGCT TCCCGGGTAG TAGTATATAC TATCCAGACT AACCCTAATT 9361 CAATAGCATA TGTTACCCAA CGGGAAGCAT ATGCTATCGA ATTAGGGTTA GTAAAAGGGT 9421 CCTAAGGAAC AGCGATATCT CCCACCCCAT GAGCTGTCAC GGTTTTATTT ACATGGGGTC 9481 AGGATTCCAC GAGGGTAGTG AACCATTTTA GTCACAAGGG CAGTGGCTGA AGATCAAGGA 9541 GCGGGCAGTG AACTCTCCTG AATCTTCGCC TGCTTCTTCA TTCTCCTTCG TTTAGCTAAT 9601 AGAATAACTG CTGAGTTGTG AACAGTAAGG TGTATGTGAG GTGCTCGAAA ACAAGGTTTC 9661 AGGTGACGCC CCCAGAATAA AATTTGGACG GGGGGTTCAG TGGTGGCATT GTGCTATGAC 9721 ACCAATATAA CCCTCACAAA CCCCTTGGGC AATAAATACT AGTGTAGGAA TGAAACATTC 9781 TGAATATCTT TAACAATAGA AATCCATGGG GTGGGGACAA GCCGTAAAGA CTGGATGTCC 9841 ATCTCACACG AATTTATGGC TATGGGCAAC ACATAATCCT AGTGCAATAT GATACTGGGG 9901 TTATTAAGAT GTGTCCCAGG CAGGGACCAA GACAGGTGAA CCATGTTGTT ACACTCTATT 9961 TGTAACAAGG GGAAAGAGAG TGGACGCCGA CAGCAGCGGA CTCCACTGGT TGTCTCTAAC 10021 ACCCCCGAAA ATTAAACGGG GCTCCACGCC AATGGGGCCC ATAAACAAAG ACAAGTGGCC 10081 ACTCTTTTTT TTGAAATTGT GGAGTGGGGG CACGCGTCAG CCCCCACACG CCGCCCTGCG 10141 GTTTTGGACT GTAAAATAAG GGTGTAATAA CTTGGCTGAT TGTAACCCCG CTAACCACTG 10201 CGGTCAAACC ACTTGCCCAC AAAACCACTA ATGGCACCCC GGGGAATACC TGCATAAGTA 10261 GGTGGGCGGG CCAAGATAGG GGCGCGATTG CTGCGATCTG GAGGACAAAT TACACACACT 10321 TGCGCCTGAG CGCCAAGCAC AGGGTTGTTG GTCCTCATAT TCACGAGGTC GCTGAGAGCA 10381 CGGTGGGCTA ATGTTGCCAT GGGTAGCATA TACTACCCAA ATATCTGGAT AGCATATGCT 10441 ATCCTAATCT ATATCTGGGT AGCATAGGCT ATCCTAATCT ATATCTGGGT AGCATATGCT 10501 ATCCTAATCT ATATCTGGGT AGTATATGCT ATCCTAATTT ATATCTGGGT AGCATAGGCT 10561 ATCCTAATCT ATATCTGGGT AGCATATGCT ATCCTAATCT ATATCTGGGT AGTATATGCT 10621 ATCCTAATCT GTATCCGGGT AGCATATGCT ATCCTAATAG AGATTAGGGT AGTATATGCT 10681 ATCCTAATTT ATATCTGGGT AGCATATACT ACCCAAATAT CTGGATAGCA TATGCTATCC 10741 TAATCTATAT CTGGGTAGCA TATGCTATCC TAATCTATAT CTGGGTAGCA TAGGCTATCC 10801 TAATCTATAT CTGGGTAGCA TATGCTATCC TAATCTATAT CTGGGTAGTA TATGCTATCC 10861 TAATTTATAT CTGGGTAGCA TAGGCTATCC TAATCTATAT CTGGGTAGCA TATGCTATCC 10921 TAATCTATAT CTGGGTAGTA TATGCTATCC TAATCTGTAT CCGGGTAGCA TATGCTATCC 10981 TCATGCATAT ACAGTCAGCA TATGATACCC AGTAGTAGAG TGGGAGTGCT ATCCTTTGCA 11041 TATGCCGCCA CCTCCCAAGG GGGCGTGAAT TTTCGCTGCT TGTCCTTTTC CTGCATGCTG 11101 GTTGCTCCCA TTCTTAGGTG AATTTAAGGA GGCCAGGCTA AAGCCGTCGC ATGTCTGATT 11161 GCTCACCAGG TAAATGTCGC TAATGTTTTC CAACGCGAGA AGGTGTTGAG CGCGGAGCTG 11221 AGTGACGTGA CAACATGGGT ATGCCCAATT GCCCCATGTT GGGAGGACGA AAATGGTGAC 11281 AAGACAGATG GCCAGAAATA CACCAACAGC ACGCATGATG TCTACTGGGG ATTTATTCTT 11341 TAGTGCGGGG GAATACACGG CTTTTAATAC GATTGAGGGC GTCTCCTAAC AAGTTACATC 11401 ACTCCTGCCC TTCCTCACCC TCATCTCCAT CACCTCCTTC ATCTCCGTCA TCTCCGTCAT 11461 CACCCTCCGC GGCAGCCCCT TCCACCATAG GTGGAAACCA GGGAGGCAAA TCTACTCCAT 11521 CGTCAAAGCT GCACACAGTC ACCCTGATAT TGCAGGTAGG AGCGGGCTTT GTCATAACAA 11581 GGTCCTTAAT CGCATCCTTC AAAACCTCAG CAAATATATG AGTTTGTAAA AAGACCATGA 11641 AATAACAGAC AATGGACTCC CTTAGCGGGC CAGGTTGTGG GCCGGGTCCA GGGGCCATTC 11701 CAAAGGGGAG ACGACTCAAT GGTGTAAGAC GACATTGTGG AATAGCAAGG GCAGTTCCTC 11761 GCCTTAGGTT GTAAAGGGAG GTCTTACTAC CTCCATATAC GAACACACCG GCGACCCAAG 11821 TTCCTTCGTC GGTAGTCCTT TCTACGTGAC TCCTAGCCAG GAGAGCTCTT AAACCTTCTG 11881 CAATGTTCTC AAATTTCGGG TTGGAACCTC CTTGACCACG ATGCTTTCCA AACCACCCTC 11941 CTTTTTTGCG CCTGCCTCCA TCACCCTGAC CCCGGGGTCC AGTGCTTGGG CCTTCTCCTG 12001 GGTCATCTGC GGGGCCCTGC TCTATCGCTC CCGGGGGCAC GTCAGGCTCA CCATCTGGGC 12061 CACCTTCTTG GTGGTATTCA AAATAATCGG CTTCCCCTAC AGGGTGGAAA AATGGCCTTC 12121 TACCTGGAGG GGGCCTGCGC GGTGGAGACC CGGATGATGA TGACTGACTA CTGGGACTCC 12181 TGGGCCTCTT TTCTCCACGT CCACGACCTC TCCCCCTGGC TCTTTCACGA CTTCCCCCCC 12241 TGGCTCTTTC ACGTCCTCTA CCCCGGCGGC CTCCACTACC TCCTCGACCC CGGCCTCCAC 12301 TACCTCCTCG ACCCCGGCCT CCACTGCCTC CTCGACCCCG GCCTCCACCT CCTGCTCCTG 12361 CCCCTCCTGC TCCTGCCCCT CCTCCTGCTC CTGCCCCTCC TGCCCCTCCT GCTCCTGCCC 12421 CTCCTGCCCC TCCTGCTCCT GCCCCTCCTG CCCCTCCTGC TCCTGCCCCT CCTGCCCCTC 12481 CTCCTGCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC CCCTCCTGCC CCTCCTGCTC 12541 CTGCCCCTCC TGCCCCTCCT GCTCCTGCCC CTCCTGCCCC TCCTGCTCCT GCCCCTCCTG 12601 CTCCTGCCCC TCCTGCTCCT GCCCCTCCTG CTCCTGCCCC TCCTGCCCCT CCTGCCCCTC 12661 CTCCTGCTCC TGCCCCTCCT GCTCCTGCCC CTCCTGCCCC TCCTGCCCCT CCTGCTCCTG 12721 CCCCTCCTCC TGCTCCTGCC CCTCCTGCCC CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC 12781 CTGCCCCTCC TCCTGCTCCT GCCCCTCCTC CTGCTCCTGC CCCTCCTGCC CCTCCTGCCC 12841 CTCCTCCTGC TCCTGCCCCT CCTGCCCCTC CTCCTGCTCC TGCCCCTCCT CCTGCTCCTG 12901 CCCCTCCTGC CCCTCCTGCC CCTCCTCCTG CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC 12961 CTGCCCCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC CCCTCCTCCT GCTCCTGCCC 13021 CTCCTGCTCC TGCCCCTCCC GCTCCTGCTC CTGCTCCTGT TCCACCGTGG GTCCCTTTGC 13081 AGCCAATGCA ACTTGGACGT TTTTGGGGTC TCCGGACACC ATCTCTATGT CTTGGCCCTG 13141 ATCCTGAGCC GCCCGGGGCT CCTGGTCTTC CGCCTCCTCG TCCTCGTCCT CTTCCCCGTC 13201 CTCGTCCATG GTTATCACCC CCTCTTCTTT GAGGTCCACT GCCGCCGGAG CCTTCTGGTC 13261 CAGATGTGTC TCCCTTCTCT CCTAGGCCAT TTCCAGGTCC TGTACCTGGC CCCTCGTCAG 13321 ACATGATTCA CACTAAAAGA GATCAATAGA CATCTTTATT AGACGACGCT CAGTGAATAC 13381 AGGGAGTGCA GACTCCTGCC CCCTCCAACA GCCCCCCCAC CCTCATCCCC TTCATGGTCG 13441 CTGTCAGACA GATCCAGGTC TGAAAATTCC CCATCCTCCG AACCATCCTC GTCCTCATCA 13501 CCAATTACTC GCAGCCCGGA AAACTCCCGC TGAACATCCT CAAGATTTGC GTCCTGAGCC 13561 TCAAGCCAGG CCTCAAATTC CTCGTCCCCC TTTTTGCTGG ACGGTAGGGA TGGGGATTCT 13621 CGGGACCCCT CCTCTTCCTC TTCAAGGTCA CCAGACAGAG ATGCTACTGG GGCAACGGAA 13681 GAAAAGCTGG GTGCGGCCTG TGAGGATCAG CTTATCGATG ATAAGCTGTC AAACATGAGA 13741 ATTCTTGAAG ACGAAAGGGC CTCGTGATAC GCCTATTTTT ATAGGTTAAT GTCATGATAA 13801 TAATGGTTTC TTAGACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT 13861 GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA 13921 TGCTTCAATA ATATTGAAAA AGGAAGAGTA TGAGTATTCA ACATTTCCGT GTCGCCCTTA 13981 TTCCCTTTTT TGCGGCATTT TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG 14041 TAAAAGATGC TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA 14101 GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG AAGAACGTTT TCCAATGATG AGCACTTTTA 14161 AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTGTTGACGC CGGGCAAGAG CAACTCGGTC 14221 GCCGCATACA CTATTCTCAG AATGACTTGG TTGAGTACTC ACCAGTCACA GAAAAGCATC 14281 TTACGGATGG CATGACAGTA AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA 14341 CTGCGGCCAA CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC 14401 ACAACATGGG GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG AATGAAGCCA 14461 TACCAAACGA CGAGCGTGAC ACCACGATGC CTGCAGCAAT GGCAACAACG TTGCGCAAAC 14521 TATTAACTGG CGAACTACTT ACTCTAGCTT CCCGGCAACA ATTAATAGAC TGGATGGAGG 14581 CGGATAAAGT TGCAGGACCA CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG 14641 ATAAATCTGG AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG 14701 GTAAGCCCTC CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT ATGGATGAAC 14761 GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA GCATTGGTAA CTGTCAGACC 14821 AAGTTTACTC ATATATACTT TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT 14881 AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC 14941 ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC 15001 GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG 15061 ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA 15121 ATACTGTCCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC 15181 CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT 15241 GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA 15301 CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC 15361 TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC 15421 CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT 15481 GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT 15541 GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC 15601 TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG 15661 ATAACCGTAT TACCGCCTTT GAGTGAGCTG ATACCGCTCG CCGCAGCCGA ACGACCGAGC 15721 GCAGCGAGTC AGTGAGCGAG GAAGCGGAAG AGCGCCTGAT GCGGTATTTT CTCCTTACGC 15781 ATCTGTGCGG TATTTCACAC CGCATATGGT GCACTCTCAG TACAATCTGC TCTGATGCCG 15841 CATAGTTAAG CCAGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC 15901 CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT 15961 CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA 16021 TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC 16081 CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG 16141 AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTTG 16201 CATGCCTGCA GGTCGGCCGC CACGACCGGT GCCGCCACCA TCCCCTGACC CACGCCCCTG 16261 ACCCCTCACA AGGAGACGAC CTTCCATGAC CGAGTACAAG CCCACGGTGC GCCTCGCCAC 16321 CCGCGACGAC GTCCCCCGGG CCGTACGCAC CCTCGCCGCC GCGTTCGCCG ACTACCCCGC 16381 CACGCGCCAC ACCGTCGACC CGGACCGCCA CATCGAGCGG GTCACCGAGC TGCAAGAACT 16441 CTTCCTCACG CGCGTCGGGC TCGACATCGG CAAGGTGTGG GTCGCGGACG ACGGCGCCGC 16501 GGTGGCGGTC TGGACCACGC CGGAGAGCGT CGAAGCGGGG GCGGTGTTCG CCGAGATCGG 16561 CCCGCGCATG GCCGAGTTGA GCGGTTCCCG GCTGGCCGCG CAGCAACAGA TGGAAGGCCT 16621 CCTGGCGCCG CACCGGCCCA AGGAGCCCGC GTGGTTCCTG GCCACCGTCG GCGTCTCGCC 16681 CGACCACCAG GGCAAGGGTC TGGGCAGCGC CGTCGTGCTC CCCGGAGTGG AGGCGGCCGA 16741 GCGCGCCGGG GTGCCCGCCT TCCTGGAGAC CTCCGCGCCC CGCAACCTCC CCTTCTACGA 16801 GCGGCTCGGC TTCACCGTCA CCGCCGACGT CGAGGTGCCC GAAGGACCGC GCACCTGGTG 16861 CATGACCCGC AAGCCCGGTG CCTGACGCCC GCCCCACGAC CCGCAGCGCC CGACCGAAAG 16921 GAGCGCACGA CCCCATGGCT CCGACCGAAG CCGACCCGGG CGGCCCCGCC GACCCCGCAC 16981 CCGCCCCCGA GGCCCACCGA CTCTAGAGGA TCATAATCAG CCATACCACA TTTGTAGAGG 17041 TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG 17101 CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA 17161 TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC 17221 TCATCAATGT ATCTTATCAT GTCTGGATCA CTCGCCGATA GTGGAAACCG ACGCCCCAGC 17281 ACTCGTCCGA GGGCAAAGGA ATAGGGGAGA TGGGGGAGGC TAACTGAAAC ACGGAAGGAG 17341 ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA GACAGAATAA AACGCACGGG 17401 TGTTGGGTCG TTTGTTCATA AACGCGGGGT TCGGTCCCAG GGCTGGCACT CTGTCGATAC 17461 CCCACCGAGA CCCCATTGGG GCCAATACGC CCGCGTTTCT TCCTTTTCCC CACCCCACCC 17521 CCCAAGTTCG GGTGAAGGCC CAGGGCTCGC AGCCAACGTC GGGGCGGCAG GCCCTGCCAT 17581 AGCCACTGGC CCCGTGGGTT AGGGACGGGG TCCCCCATGG GGAATGGTTT ATGGTTCGTG 17641 GGGGTTATTA TTTTGGGCGT TGCGTGGGGT CTGGTCCACG ACTGGACTGA GCAGACAGAC 17701 CCATGGTTTT TGGATGGCCT GGGCATGGAC CGCATGTACT GGCGCGACAC GAACACCGGG 17761 CGTCTGTGGC TGCCAAACAC CCCCGACCCC CAAAAACCAC CGCGCGGATT TCTGGCGTGC 17821 CAAGCTAGTC GACCAATTCT CATGTTTGAC AGCTTATCAT CGCAGATCCG GGCAACGTTG 17881 TTGCATTGCT GCAGGCGCAG AACTGGTAGG TATGGAAGAT CTCTAGAAGC TGGGTACCAG 17941 CTGCTAGCAA GCTTGCTAGC GGCCGGCTCG AGTTTACTCC CTATCAGTGA TAGAGAACGT 18001 ATGTCGAGTT TACTCCCTAT CAGTGATAGA GAACGATGTC GAGTTTACTC CCTATCAGTG 18061 ATAGAGAACG TATGTCGAGT TTACTCCCTA TCAGTGATAG AGAACGTATG TCGAGTTTAC 18121 TCCCTATCAG TGATAGAGAA CGTATGTCGA GTTTATCCCT ATCAGTGATA GAGAACGTAT 18181 GTCGAGTTTA CTCCCTATCA GTGATAGAGA ACGTATGTCG AGGTAGGCGT GTACGGTGGG 18241 AGGCCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT CGCCG (SEQ ID NO: 37) LINE 1-GFP mRNA (SEQ ID NO: 38) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGCCG TCAGACCATC AAGACTAGGA 1081 AGAAACTGCA TCAACTAATG AGCAAAATCA CCAGCTAACA TCATAGTATA CATGACCGGC 1141 TCTAACTCAC ATATCACCAT CCTTACACTT AACATTAACG GCCTCAACTC AGCTATCAAG 1201 CGCCATCGGC TGGCCAGCTG GATCAAATCA CAGGATCCAA GCGTTTGTTG CATCCAAGAG 1261 ACCCACCTGA CCTGTAGAGA TACTCACCGC CTCAAGATCA AGGGATGGCG AAAGATTTAT 1321 CAGGCGAACG GTAAGCAGAA GAAAGCCGGA GTCGCAATTC TGGTCTCAGA CAAGACGGAT 1381 TTCAAGCCCA CCAAAATTAA GCGTGATAAG GAAGGTCACT ATATTATGGT GAAAGGCAGC 1441 ATACAGCAGG AAGAACTTAC CATATTGAAC ATCTACGCGC CAAACACCGG CGCACCTCGC 1501 TTTATCAAAC AGGTCCTGTC CGATCTGCAG CGAGATCTGG ATTCTCATAC GTTGATTATG 1561 GGTGATTTCA ATACACCATT GAGCACCCTG GATCGCAGCA CCAGGCAAAA GGTAAATAAA 1621 GACACGCAAG AGCTCAATAG CGCACTGCAT CAGGCAGATC TCATTGATAT TTATCGCACT 1681 CTTCATCCTA AGAGTACCGA GTACACATTC TTCAGCGCCC CACATCATAC ATACTCAAAG 1741 ATCGATCATA TCGTCGGCTC AAAGGCTCTG CTGTCAAAGT GCAAGCGCAC AGAGATAATT 1801 ACAAATTACC TGTCAGATCA TAGCGCGATC AAGCTCGAGC TGAGAATCAA GAACCTGACC 1861 CAGAGCCGGA GTACCACTTG GAAGCTTAAT AACCTGCTGC TCAACGATTA TTGGGTCCAC 1921 AATGAGATGA AGGCAGAGAT TAAAATGTTC TTCGAAACAA ATGAGAATAA GGATACTACC 1981 TATCAAAACC TTTGGGATGC CTTTAAGGCC GTCTGCAGAG GCAAGTTCAT CGCCCTCAAC 2041 GCCTATAAAA GAAAACAAGA GAGATCTAAG ATCGATACTC TCACCTCTCA GCTGAAGGAG 2101 TTGGAGAAAC AGGAACAGAC CCACTCCAAG GCGTCAAGAC GGCAGGAGAT CACAAAGATT 2161 CGCGCCGAGT TGAAAGAGAT CGAAACCCAA AAGACTCTTC AGAAAATTAA CGAGTCTCGT 2221 AGTTGGTTCT TCGAGCGGAT TAATAAGATA GACAGACCTC TGGCACGACT GATTAAGAAG 2281 AAGCGCGAAA AGAACCAGAT TGATACCATC AAGAACGACA AGGGCGACAT CACTACTGAC 2341 CCGACCGAGA TCCAGACCAC TATTCGGGAG TATTATAAGC ATTTGTATGC TAACAAGCTT 2401 GAGAACCTGG AAGAGATGGA CACTTTTCTG GATACCTATA CTCTGCCACG GCTTAATCAA 2461 GAGGAAGTCG AGTCCCTCAA CCGCCCAATT ACAGGAAGCG AGATTGTGGC CATAATTAAC 2521 TCCCTGCCGA CAAAGAAATC TCCTGGTCCG GACGGGTTTA CAGCTGAGTT TTATCAACGG 2581 TATATGGAAG AGCTTGTACC GTTTCTGCTC AAGCTCTTTC AGTCTATAGA AAAGGAAGGC 2641 ATCTTGCCCA ATTCCTTCTA CGAAGCTTCT ATAATACTTA TTCCCAAACC AGGACGCGAT 2701 ACCACAAAGA AGGAAAACTT CCGGCCCATT AGTCTCATGA ATATCGACGC TAAAATATTG 2761 AACAAGATTC TCGCCAACAG AATCCAACAA CATATTAAGA AATTGATACA TCACGACCAG 2821 GTGGGGTTTA TACCTGGCAT GCAGGGCTGG TTTAACATCC GGAAGAGTAT TAACGTCATT 2881 CAACACATTA ATAGAGCTAA GGATAAGAAT CATATGATCA TCTCTATAGA CGCGGAAAAG 2941 GCATTCGATA AGATTCAGCA GCCATTTATG CTCAAGACTC TGAACAAACT CGGCATCGAC 3001 GGAACATATT TTAAGATTAT TCGCGCAATT TACGATAAGC CGACTGCTAA CATTATCCTT 3061 AACGGCCAAA AGCTCGAGGC CTTTCCGCTC AAGACTGGAA CCCGCCAAGG CTGTCCCCTC 3121 TCCCCGCTTT TGTTTAATAT TGTACTCGAG GTGCTGGCTA GGGCTATTCG TCAAGAGAAA 3181 GAGATTAAAG GGATACAGCT CGGGAAGGAA GAGGTCAAGC TTTCCTTGTT CGCCGATGAT 3241 ATGATTGTGT ACCTGGAGAA TCCTATTGTG TCTGCTCAGA ACCTTCTTAA ACTTATTTCT 3301 AACTTTAGCA AGGTCAGCGG CTATAAGATT AACGTCCAGA AATCTCAGGC CTTTCTGTAC 3361 ACAAATAATC GACAGACCGA ATCCCAGATA ATGGGTGAGC TTCCGTTTGT CATAGCCAGC 3421 AAAAGGATAA AGTATCTCGG AATCCAGCTG ACACGAGACG TTAAAGATTT GTTTAAGGAA 3481 AATTACAAGC CTCTCCTGAA AGAGATTAAG GAAGATACTA ATAAGTGGAA GAATATCCCC 3541 TGTTCATGGG TTGGCAGAAT CAACATAGTG AAGATGGCAA TACTTCCTAA AGTGATATAT 3601 CGCTTTAACG CCATCCCAAT TAAACTGCCT ATGACCTTCT TTACGGAGCT CGAGAAAACA 3661 ACCCTTAAAT TTATATGGAA TCAAAAGAGA GCAAGAATAG CGAAGTCCAT CTTGAGCCAG 3721 AAGAATAAGG CCGGTGGGAT TACTTTGCCT GATTTTAAGT TGTATTATAA AGCCACAGTA 3781 ACTAAGACAG CCTGGTATTG GTATCAGAAT AGAGACATCG ACCAGTGGAA TCGGACCGAA 3841 CCATCAGAGA TAATGCCCCA CATCTATAAT TACCTTATAT TCGATAAGCC AGAAAAGAAT 3901 AAACAGTGGG GCAAAGACAG CCTCTTCAAC AAGTGGTGTT GGGAGAATTG GCTGGCCATA 3961 TGCCGGAAAC TCAAGCTCGA CCCCTTTCTT ACACCCTACA CTAAAATCAA CAGTAGGTGG 4021 ATCAAGGACT TGAATGTCAA GCCAAAGACT ATAAAGACAC TGGAAGAGAA TCTTGGGATC 4081 ACAATACAAG ATATAGGCGT CGGCAAAGAT TTTATGTCAA AGACGCCCAA GGCCATGGCC 4141 ACTAAGGATA AGATTGATAA GTGGGACCTT ATTAAGCTCA AAAGCTTCTG TACTGCCAAG 4201 GAGACCACGA TCAGAGTTAA TAGGCAGCCC ACTACATGGG AAAAGATTTT CGCCACTTAT 4261 TCATCAGATA AGGGGTTGAT AAGCAGAATA TATAACGAGC TGAAGCAGAT CTACAAGAAG 4321 AAAACGAATA ATCCCATCAA GAAGTGGGCA AAAGATATGA ACAGGCATTT TAGCAAAGAG 4381 GATATCTACG CCGCGAAGAA GCATATGAAG AAGTGTAGTT CAAGCTTGGC CATTCGTGAG 4441 ATGCAGATTA AGACGACCAT GCGATACCAC CTTACCCCAG TGAGGATGGC AATTATCAAG 4501 AAATCTGGCA ATAATAGATG TTGGCGGGGC TGTGGCGAGA TTGGCACCCT GCTCCATTGC 4561 TGGTGGGATT GCAAGCTGGT GCAGCCGCTT TGGAAATCAG TCTGGCGCTT TCTGAGGGAC 4621 CTCGAGCTTG AGATTCCCTT CGATCCCGCA ATTCCCTTGC TCGGAATCTA TCCTAACGAA 4681 TACAAGAGCT GTTGTTACAA GGATACGTGT ACCCGGATGT TCATCGCGGC CTTGTTTACG 4741 ATAGCTAAGA CGTGGAATCA GCCTAAGTGC CCCACAATGA TCGATTGGAT CAAGAAAATG 4801 TGGCATATTT ATACCATGGA GTATTACGCA GCAATTAAGA ATGACGAATT TATTTCCTTC 4861 GTTGGGACCT GGATGAAGCT GGAGACTATT ATTCTGAGCA AGCTGTCTCA GGAGCAAAAG 4921 ACAAAGCATA GAATCTTCTC TCTCATTGGT GGTAACGACT ACAAAGACGA TGACGACAAG 4981 TAAAGCGCTT CTAGAAGTTG TCTCCTCCTG CACTGACTGA CTGATACAAT CGATTTCTGG 5041 ATCCGCAGGC CTAATCAACC TCTGGATTAC AAAATTTGTG AAAGATTGAC TGGTATTCTT 5101 AACTATGTTG CTCCTTTTAC GCTATGTGGA TACGCTGCTT TAATGCCTTT GTATCATGCT 5161 ATTGCTTCCC GTATGGCTTT CATTTTCTCC TCCTTGTATA AATCCTGGTT GCTGTCTCTT 5221 TATGAGGAGT TGTGGCCCGT TGTCAGGCAA CGTGGCGTGG TGTGCACTGT GTTTGCTGAC 5281 GCAACCCCCA CTGGTTGGGG CATTGCCACC ACCTGTCAGC TCCTTTCCGG GACTTTCGCT 5341 TTCCCCCTCC CTATTGCCAC GGCGGAACTC ATCGCCGCCT GCCTTGCCCG CTGCTGGACA 5401 GGGGCTCGGC TGTTGGGCAC TGACAATTCC GTGGTGTTGT CGGGGAAGCT GACGTCCTTT 5461 CCATGGCTGC TCGCCTGTGT TGCCACCTGG ATTCTGCGCG GGACGTCCTT CTGCTACGTC 5521 CCTTCGGCCC TCAATCCAGC GGACCTTCCT TCCCGCTGAG AGACACAAAA AATTCCAACA 5581 CACTATTGCA ATGAAAATAA ATTTCCTTTA TTAGCCAGAA GTCAGATGCT CAAGGGGCTT 5641 CATGATGTCC CCATAATTTT TGGCAGAGGG AAAAAGATCT CAGTGGTATT TGTGAGCCAG 5701 GGCATTGGCC TTCTGATAGG CAGCCTGCAC CTGAGGAGTG CGGCCGCTTT ACTTGTACAG 5761 CTCGTCCATG CCGAGAGTGA TCCCGGCGGC GGTCACGAAC TCCAGCAGGA CCATGTGATC 5821 GCGCTTCTCG TTGGGGTCTT TGCTCAGGGC GGACTGGGTG CTCAGGTAGT GGTTGTCGGG 5881 CAGCAGCACG GGGCCGTCGC CGATGGGGGT GTTCTGCTGG TAGTGGTCGG CGAGCTGCAC 5941 GCTGCCGTCC TCGATGTTGT GGCGGATCTT GAAGTTCACC TTGATGCCGT TCTTCTGCTT 6001 GTCGGCCATG ATATAGACGT TGTGGCTGTT GTAGTTGTAC TCCAGCTTGT GCCCCAGGAT 6061 GTTGCCGTCC TCCTTGAAGT CGATGCCCTT CAGCTCGATG CGGTTCACCA GGGTGTCGCC 6121 CTCGAACTTC ACCTCGGCGC GGGTCTTGTA GTTGCCGTCG TCCTTGAAGA AGATGGTGCG 6181 CTCCTGGACG TAGCCTTCGG GCATGGCGGA CTTGAAGAAG TCGTGCTGCT TCATGTGGTC 6241 GGGGTAGCGG CTGAAGCACT GCACGCCGTA GGTCAGGGTG GTCACGAGGG TGGGCCAGGG 6301 CACGGGCAGC TTGCCGGTGG TGCAGATGAA CTTCAGGGTC AGCTTGCCGT AGGTGGCATC 6361 GCCCTCGCCC TCGCCGGACA CGCTGAACTT GTGGCCGTTT ACGTCGCCGT CCAGCTCGAC 6421 CAGGATGGGC ACCACCCCGG TGAACAGCTC CTCGCCCTTG CTCACCATGG TGGCGGGATC 6481 TGACGGTTCA CTAAACCAGC TCTGCTTATA TAGACCTCCC ACCGTACACG CCTACCGCCC 6541 ATTTGCGTCA ATGGGGCGGA GTTGTTACGA CATTTTGGAA AGTCCCGTTG ATTTTGGTGC 6601 CAAAACAAAC TCCCATTGAC GTCAATGGGG TGGAGACTTG GAAATCCCCG TGAGTCAAAC 6661 CGCTATCCAC GCCCATTGAT GTACTGCCAA AACCGCATCA CCATGGTAAT AGCGATGACT 6721 AATACGTAGA TGTACTGCCA AGTAGGAAAG TCCCATAAGG TCATGTACTG GGCATAATGC 6781 CAGGCGGGCC ATTTACCGTC ATTGACGTCA ATAGGGGGCG TACTTGGCAT ATGATACACT 6841 TGATGTACTG CCAAGTGGGC AGTTTACCGT AAATACTCCA CCCATTGACG TCAATGGAAA 6901 GTCCCTATTG GCGTTACTAT GGGAACATAC GTCATTATTG ACGTCAATGG GCGGGGGTCG 6961 TTGGGCGGTC AGCCAGGCGG GCCATTTACC GTAAGTTATG TAACGGGCCT GCTGCCGGCT 7021 CTGCGGCCTC TTCCGCGTCT TCGCCTTCGC CCTCAGACGA GTCGGATCTC CCTTTGGGCC 7081 GCCTCCCCGC CTGTCTAGCT TGACTGACTG AGATACAGCG TACCTTCAGC TCACAGACAT 7141 GATAAGATAC ATTGATGAGT TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT 7201 TATTTGTGAA ATTTGTGATG CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA 7261 AGTT (SEQ ID NO: 38) LINE-1-plasmid_CD5-intron-FCR-PI3K (SEQ ID NO: 39) 1 CGGCCGCGGG GGGAGGAGCC AAGATGGCCG AATAGGAACA GCTCCGGTCT ACAGCTCCCA 61 GCGTGAGCGA CGCAGAAGAC GGTGATTTCT GCATTTCCAT CTGAGGTACC GGGTTCATCT 121 CACTAGGGAG TGCCAGACAG TGGGCGCAGG CCAGTGTGTG TGCGCACCGT GCGCGAGCCG 181 AAGCAGGGCG AGGCATTGCC TCACCTGGGA AGCGCAAGGG GTCAGGGAGT TCCCTTTCCG 241 AGTCAAAGAA AGGGGTGACG GACGCACCTG GAAAATCGGG TCACTCCCAC CCGAATATTG 301 CGCTTTTCAG ACCGGCTTAA GAAACGGCGC ACCACGAGAC TATATCCCAC ACCTGGCTCG 361 GAGGGTCCTA CGCCCACGGA ATCTCGCTGA TTGCTAGCAC AGCAGTCTGA GATCAAACTG 421 CAAGGCGGCA ACGAGGCTGG GGGAGGGGCG CCCGCCATTG CCCAGGCTTG CTTAGGTAAA 481 CAAAGCAGCA GGGAAGCTCG AACTGGGTGG AGCCCACCAC AGCTCAAGGA GGCCTGCCTG 541 CCTCTGTAGG CTCCACCTCT GGGGGCAGGG CACAGACAAA CAAAAAGACA GCAGTAACCT 601 CTGCAGACTT AAGTGTCCCT GTCTGACAGC TTTGAAGAGA GCAGTGGTTC TCCCAGCACG 661 CAGCTGGAGA TCTGAGAACG GGCAGACTGC CTCCTCAAGT GGGTCCCTGA CCCCTGACCC 721 CCGAGCAGCC TAACTGGGAG GCACCCCCCA GCAGGGGCAC ACTGACACCT CACACGGCAG 781 GGTATTCCAA CAGACCTGCA GCTGAGGGTC CTGTCTGTTA GAAGGAAAAC TAACAACCAG 841 AAAGGACATC TACACCGAAA ACCCATCTGT ACATCACCAT CATCAAAGAC CAAAAGTAGA 901 TAAAACCACA AAGATGGGGA AAAAACAGAA CAGAAAAACT GGAAACTCTA AAACGCAGAG 961 CGCCTCTCCT CCTCCAAAGG AACGCAGTTC CTCACCAGCA ACAGAACAAA GCTGGATGGA 1021 GAATGATTTT GATGAGCTGA GAGAAGAAGG CTTCAGACGA TCAAATTACT CTGAGCTACG 1081 GGAGGACATT CAAACCAAAG GCAAAGAAGT TGAAAACTTT GAAAAAAATT TAGAAGAATG 1141 TATAACTAGA ATAACCAATA CAGAGAAGTG CTTAAAGGAG CTGATGGAGC TGAAAACCAA 1201 GGCTCGAGAA CTACGTGAAG AATGCAGAAG CCTCAGGAGC CGATGCGATC AACTGGAAGA 1261 AAGGGTATCA GCAATGGAAG ATGAAATGAA TGAAATGAAG CGAGAAGGGA AGTTTAGAGA 1321 AAAAAGAATA AAAAGAAATG AGCAAAGCCT CCAAGAAATA TGGGACTATG TGAAAAGACC 1381 AAATCTACGT CTGATTGGTG TACCTGAAAG TGATGTGGAG AATGGAACCA AGTTGGAAAA 1441 CACTCTGCAG GATATTATCC AGGAGAACTT CCCCAATCTA GCAAGGCAGG CCAACGTTCA 1501 GATTCAGGAA ATACAGAGAA CGCCACAAAG ATACTCCTCG AGAAGAGCAA CTCCAAGACA 1561 CATAATTGTC AGATTCACCA AAGTTGAAAT GAAGGAAAAA ATGTTAAGGG CAGCCAGAGA 1621 GAAAGGTCGG GTTACCCTCA AAGGAAAGCC CATCAGACTA ACAGCGGATC TCTCGGCAGA 1681 AACCCTACAA GCCAGAAGAG AGTGGGGGCC AATATTCAAC ATTCTTAAAG AAAAGAATTT 1741 TCAACCCAGA ATTTCATATC CAGCCAAACT AAGCTTCATA AGTGAAGGAG AAATAAAATA 1801 CTTTATAGAC AAGCAAATGT TGAGAGATTT TGTCACCACC AGGCCTGCCC TAAAAGAGCT 1861 CCTGAAGGAA GCGCTAAACA TGGAAAGGAA CAACCGGTAC CAGCCGCTGC AAAATCATGC 1921 CAAAATGTAA AGACCATCAA GACTAGGAAG AAACTGCATC AACTAATGAG CAAAATCACC 1981 AGCTAACATC ATAATGACAG GATCAACTTC ACACATAACA ATATTAACTT TAAATATAAA 2041 TGGACTAAAT TCTGCAATTA AAAGACACAG ACTGGCAAGT TGGATAAAGA GTCAAGACCC 2101 ATCAGTGTGC TGTATTCAGG AAACCCATCT CACGTGCAGA GACACACATA GGCTCAAAAT 2161 AAAAGGATGG AGGAAGATCT ACCAAGCCAA TGGAAAACAA AAAAAGGCAG GGGTTGCAAT 2221 CCTAGTCTCT GATAAAACAG ACTTTAAACC AACAAAGATC AAAAGAGACA AAGAAGGCCA 2281 TTACATAATG GTAAAGGGAT CAATTCAACA AGAGGAGCTA ACTATCCTAA ATATTTATGC 2341 ACCCAATACA GGAGCACCCA GATTCATAAA GCAAGTCCTC AGTGACCTAC AAAGAGACTT 2401 AGACTCCCAC ACATTAATAA TGGGAGACTT TAACACCCCA CTGTCAACAT TAGACAGATC 2461 AACGAGACAG AAAGTCAACA AGGATACCCA GGAATTGAAC TCAGCTCTGC ACCAAGCAGA 2521 CCTAATAGAC ATCTACAGAA CTCTCCACCC CAAATCAACA GAATATACAT TTTTTTCAGC 2581 ACCACACCAC ACCTATTCCA AAATTGACCA CATAGTTGGA AGTAAAGCTC TCCTCAGCAA 2641 ATGTAAAAGA ACAGAAATTA TAACAAACTA TCTCTCAGAC CACAGTGCAA TCAAACTAGA 2701 ACTCAGGATT AAGAATCTCA CTCAAAGCCG CTCAACTACA TGGAAACTGA ACAACCTGCT 2761 CCTGAATGAC TACTGGGTAC ATAACGAAAT GAAGGCAGAA ATAAAGATGT TCTTTGAAAC 2821 CAACGAGAAC AAAGACACCA CATACCAGAA TCTCTGGGAC GCATTCAAAG CAGTGTGTAG 2881 AGGGAAATTT ATAGCACTAA ATGCCTACAA GAGAAAGCAG GAAAGATCCA AAATTGACAC 2941 CCTAACATCA CAATTAAAAG AACTAGAAAA GCAAGAGCAA ACACATTCAA AAGCTAGCAG 3001 AAGGCAAGAA ATAACTAAAA TCAGAGCAGA ACTGAAGGAA ATAGAGACAC AAAAAACCCT 3061 TCAAAAAATC AATGAATCCA GGAGCTGGTT TTTTGAAAGG ATCAACAAAA TTGATAGACC 3121 GCTAGCAAGA CTAATAAAGA AAAAAAGAGA GAAGAATCAA ATAGACACAA TAAAAAATGA 3181 TAAAGGGGAT ATCACCACCG ATCCCACAGA AATACAAACT ACCATCAGAG AATACTACAA 3241 ACACCTCTAC GCAAATAAAC TAGAAAATCT AGAAGAAATG GATACATTCC TCGACACATA 3301 CACTCTCCCA AGACTAAACC AGGAAGAAGT TGAATCTCTG AATCGACCAA TAACAGGCTC 3361 TGAAATTGTG GCAATAATCA ATAGTTTACC AACCAAAAAG AGTCCAGGAC CAGATGGATT 3421 CACAGCCGAA TTCTACCAGA GGTACAAGGA GGAACTGGTA CCATTCCTTC TGAAACTATT 3481 CCAATCAATA GAAAAAGAGG GAATCCTCCC TAACTCATTT TATGAGGCCA GCATCATTCT 3541 GATACCAAAG CCGGGCAGAG ACACAACCAA AAAAGAGAAT TTTAGACCAA TATCCTTGAT 3601 GAACATTGAT GCAAAAATCC TCAATAAAAT ACTGGCAAAC CGAATCCAGC AGCACATCAA 3661 AAAGCTTATC CACCATGATC AAGTGGGCTT CATCCCTGGG ATGCAAGGCT GGTTCAATAT 3721 ACGCAAATCA ATAAATGTAA TCCAGCATAT AAACAGAGCC AAAGACAAAA ACCACATGAT 3781 TATCTCAATA GATGCAGAAA AAGCCTTTGA CAAAATTCAA CAACCCTTCA TGCTAAAAAC 3841 TCTCAATAAA TTAGGTATTG ATGGGACGTA TTTCAAAATA ATAAGAGCTA TCTATGACAA 3901 ACCCACAGCC AATATCATAC TGAATGGGCA AAAACTGGAA GCATTCCCTT TGAAAACCGG 3961 CACAAGACAG GGATGCCCTC TCTCACCGCT CCTATTCAAC ATAGTGTTGG AAGTTCTGGC 4021 CAGGGCAATC AGGCAGGAGA AGGAAATAAA GGGTATTCAA TTAGGAAAAG AGGAAGTCAA 4081 ATTGTCCCTG TTTGCAGACG ACATGATTGT TTATCTAGAA AACCCCATCG TCTCAGCCCA 4141 AAATCTCCTT AAGCTGATAA GCAACTTCAG CAAAGTCTCA GGATACAAAA TCAATGTACA 4201 AAAATCACAA GCATTCTTAT ACACCAACAA CAGACAAACA GAGAGCCAAA TCATGGGTGA 4261 ACTCCCATTC ACAATTGCTT CAAAGAGAAT AAAATACCTA GGAATCCAAC TTACAAGGGA 4321 TGTGAAGGAC CTCTTCAAGG AGAACTACAA ACCACTGCTC AAGGAAATAA AAGAGGAGAC 4381 AAACAAATGG AAGAACATTC CATGCTCATG GGTAGGAAGA ATCAATATCG TGAAAATGGC 4441 CATACTGCCC AAGGTAATTT ACAGATTCAA TGCCATCCCC ATCAAGCTAC CAATGACTTT 4501 CTTCACAGAA TTGGAAAAAA CTACTTTAAA GTTCATATGG AACCAAAAAA GAGCCCGCAT 4561 TGCCAAGTCA ATCCTAAGCC AAAAGAACAA AGCTGGAGGC ATCACACTAC CTGACTTCAA 4621 ACTATACTAC AAGGCTACAG TAACCAAAAC AGCATGGTAC TGGTACCAAA ACAGAGATAT 4681 AGATCAATGG AACAGAACAG AGCCCTCAGA AATAATGCCG CATATCTACA ACTATCTGAT 4741 CTTTGACAAA CCTGAGAAAA ACAAGCAATG GGGAAAGGAT TCCCTATTTA ATAAATGGTG 4801 CTGGGAAAAC TGGCTAGCCA TATGTAGAAA GCTGAAACTG GATCCCTTCC TTACACCTTA 4861 TACAAAAATC AATTCAAGAT GGATTAAAGA TTTAAACGTT AAACCTAAAA CCATAAAAAC 4921 CCTAGAAGAA AACCTAGGCA TTACCATTCA GGACATAGGC GTGGGCAAGG ACTTCATGTC 4981 CAAAACACCA AAAGCAATGG CAACAAAAGA CAAAATTGAC AAATGGGATC TAATTAAACT 5041 AAAGAGCTTC TGCACAGCAA AAGAAACTAC CATCAGAGTG AACAGGCAAC CTACAACATG 5101 GGAGAAAATT TTTGCAACCT ACTCATCTGA CAAAGGGCTA ATATCCAGAA TCTACAATGA 5161 ACTCAAACAA ATTTACAAGA AAAAAACAAA CAACCCCATC AAAAAGTGGG CGAAGGACAT 5221 GAACAGACAC TTCTCAAAAG AAGACATTTA TGCAGCCAAA AAACACATGA AGAAATGCTC 5281 ATCATCACTG GCCATCAGAG AAATGCAAAT CAAAACCACT ATGAGATATC ATCTCACACC 5341 AGTTAGAATG GCAATCATTA AAAAGTCAGG AAACAACAGG TGCTGGAGAG GATGCGGAGA 5401 AATAGGAACA CTTTTACACT GTTGGTGGGA CTGTAAACTA GTTCAACCAT TGTGGAAGTC 5461 AGTGTGGCGA TTCCTCAGGG ATCTAGAACT AGAAATACCA TTTGACCCAG CCATCCCATT 5521 ACTGGGTATA TACCCAAATG AGTATAAATC ATGCTGCTAT AAAGACACAT GCACACGTAT 5581 GTTTATTGCG GCACTATTCA CAATAGCAAA GACTTGGAAC CAACCCAAAT GTCCAACAAT 5641 GATAGACTGG ATTAAGAAAA TGTGGCACAT ATACACCATG GAATACTATG CAGCCATAAA 5701 AAATGATGAG TTCATATCCT TTGTAGGGAC ATGGATGAAA TTGGAAACCA TCATTCTCAG 5761 TAAACTATCG CAAGAACAAA AAACCAAACA CCGCATATTC TCACTCATAG GTGGGAATTG 5821 AACAATGAGA TCACATGGAC ACAGGAAGGG GAATATCACA CTCTGGGGAC TGTGGTGGGG 5881 TCGGGGGAGG GGGGAGGGAT AGCATTGGGA GATATACCTA ATGCTAGATG ACACATTAGT 5941 GGGTGCAGCG CACCAGCATG GCACATGTAT ACGGATCCGA ATTCTCGACG GATCGATCCG 6001 AACAAACGAC CCAACACCCG TGCGTTTTAT TCTGTCTTTT TATTGCCGAT CCCCTCAGAA 6061 GAACTCGTCA AGAAGGCGAT AGAAGGCGAT GCGCTGCGAA TCGGGAGCGG CGATACCGTA 6121 AAGCACGAGG AAGCGGTCAG CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC 6181 CAACGCTATG TCCTGATAGC GGTCGGCCGC TCATGTTCTC GTAGGAGTCG GCGTCCTCTT 6241 CGTGGTTAGG TCCAGGTTGG CCTCTGATAG ACCGCAGCTG AGGAGCGGCG TACAGAATGC 6301 CTCTCATGTC CTCATAGCTG CCGCTGCCTT GTGGAGGCTT CTCGTGCTTC AGTGTCTCGT 6361 ATGTCTCTTG ATTCCGGGTG CTCAGGCCGG TGTACACGCC ATCAGATTTC TCGTAGCTGG 6421 TGATGGCGGC CTTCCGCACT TGGATCTTCA GCCGTCTGCA GTACAGGGTG ATGACCAGAG 6481 ACAGCAGCAG GACACCACAT GTGCCAGCCA GAGGGGCCCA AATGTAGATA TCCAGGCCTC 6541 TGGTATGCAC AGCTCCGCCT GCAGCAGGTC TACAGGCTTC AGGTCTGAGA GACAGAGGCT 6601 GGCTGGCGAT TGTAGGAGCT GGTGTAGGTG GTCTAGGAGC GGGTGTTGTT GTAGGCTTGG 6661 CGGGCAGAAA CACGGGCACG AAGTGGCTGA AGTACATGAT GCTATTGCTC AGGGCTCCGC 6721 TTCCTCCGCC GCCTGATTTG ATTTCCAGCT TGGTGCCTCC GCCAAATGTC CAAGGGCTCT 6781 CGTCGTACTG CTGGCAGTAG TAGATGCCGA AGTCCTCGTA CTGCAGGCTG CTGATTGTCA 6841 GGGTGTAGTC GGTGCCAGAG CCGCTGCCAG AAAATCTGCT TGGCACGCCG CTTTCCAGTC 6901 TGTTGGCCCG GTAGATCAGT GTCTTAGGGG CCTTGCCAGG CTTCTGCTGG AACCAGCTCA 6961 GGTAGCTGTT GATGTCCTGG CTGGCTCTAC AGGTGATGGT CACTCTATCG CCCACAGAGG 7021 CAGACAGGCT GCTAGGGCTC TGTGTCATCT GGATATCAGA GCCACCACCG CCAGATCCAC 7081 CGCCACCTGA TCCTCCGCCT CCGCTAGAAA CTGTCACTGT GGTGCCCTGG CCCCACACAT 7141 CGAAGTACCA GTCGTAGCCT CTTCTGGTGC AGAAGTACAC GGCGGTATCC TCGGCTCTCA 7201 GGCTGTTGAT CTGCAGGTAG GCGGTGTTCT TGCTGTCGTC CAGGCTGAAG GTGAATCTGC 7261 CCTTAAAGCT ATCGGCGTAG GTTGGCTCGC CGGTGTGGGT ATTGATCCAG CCCATCCACT 7321 CAAGGCCAGG TGAGTCCAGG AGATGTTTCA GCACTGTTGC CTTTAGTCTC GAGGCAACTT 7381 AGACAACTGA GTATTGATCT GAGCACAGCA GGGTGTGAGC TGTTTGAAGA TACTGGGGTT 7441 GGGGGTGAAG AAACTGCAGA GGACTAACTG GGCTGAGACC CAGTGGCAAT GTTTTAGGGC 7501 CTAAGGAATG CCTCTGAAAA TCTAGATGGA CAACTTTGAC TTTGAGAAAA GAGAGGTGGA 7561 AATGAGGAAA ATGACTTTTC TTTATTAGAT TTCGGTAGAA AGAACTTTCA TCTTTCCCCT 7621 ATTTTTGTTA TTCGTTTTAA AACATCTATC TGGAGGCAGG ACAAGTATGG TCATTAAAAA 7681 GATGCAGGCA GAAGGCATAT ATTGGCTCAG TCAAAGTGGG GAACTTTGGT GGCCAAACAT 7741 ACATTGCTAA GGCTATTCCT ATATCAGCTG GACACATATA AAATGCTGCT AATGCTTCAT 7801 TACAAACTTA TATCCTTTAA TTCCAGATGG GGGCAAAGTA TGTCCAGGGG TGAGGAACAA 7861 TTGAAACATT TGGGCTGGAG TAGATTTTGA AAGTCAGCTC TGTGTGTGTG TGTGTGTGTG 7921 TGTGTGTGAG AGCGTGTGTT TCTTTTAACG TTTTCAGCCT ACAGCATACA GGGTTCATGG 7981 TGGCAAGAAG ATAACAAGAT TTAAATTATG GCCAGTGACT AGTGCTGCAA GAAGAACAAC 8041 TACCTGCATT TAATGGGAAA GCAAAATCTC AGGCTTTGAG GGAAGTTAAC ATAGGCTTGA 8101 TTCTGGGTGG AAGCTGGGTG TGTAGTTATC TGGAGGCCAG GCTGGAGCTC TCAGCTCACT 8161 ATGGGTTCAT CTTTATTGTC TCCTTTTTCC AGGGGCCTGT CGGACCCAGT TCATGCCGTA 8221 GTTGGTGAAG GTGTAGCCGC TGGCGGCACA GCTGATTCTG ACAGATCCGC CAGGTTTCAC 8281 AAGTCCGCCG CCAGACTGAA CCAGCTGGAT CTCAGAGATG CTACAGGCCA CTGTTCCCAG 8341 CAGCAGCAGA GACTGCAGCC ACATCTGGTG GCGAATTCGA AGCTTGAGCT CGAGATCTGA 8401 GTCCGGTAGC GCTAGCGGAT CTGACGGTTC ACTAAACCAG CTCTGCTTAT ATAGACCTCC 8461 CACCGTACAC GCCTACCGCC CATTTGCGTC AATGGGGCGG AGTTGTTACG ACATTTTGGA 8521 AAGTCCCGTT GATTTTGGTG CCAAAACAAA CTCCCATTGA CGTCAATGGG GTGGAGACTT 8581 GGAAATCCCC GTGAGTCAAA CCGCTATCCA CGCCCATTGA TGTACTGCCA AAACCGCATC 8641 ACCATGGTAA TAGCGATGAC TAATACGTAG ATGTACTGCC AAGTAGGAAA GTCCCATAAG 8701 GTCATGTACT GGGCATAATG CCAGGCGGGC CATTTACCGT CATTGACGTC AATAGGGGGC 8761 GTACTTGGCA TATGATACAC TTGATGTACT GCCAAGTGGG CAGTTTACCG TAAATACTCC 8821 ACCCATTGAC GTCAATGGAA AGTCCCTATT GGCGTTACTA TGGGAACATA CGTCATTATT 8881 GACGTCAATG GGCGGGGGTC GTTGGGCGGT CAGCCAGGCG GGCCATTTAC CGTAAGTTAT 8941 GTAACGCGGA ACTCCATATA TGGGCTATGA ACTAATGACC CCGTAATTGA TTACTATTAG 9001 CCCGGGGGAT CCAGACATGA TAAGATACAT TGATGAGTTT GGACAAACCA CAACTAGAAT 9061 GCAGTGAAAA AAATGCTTTA TTTGTGAAAT TTGTGATGCT ATTGCTTTAT TTGTAACCAT 9121 TATAAGCTGC AATAAACAAG TTAACAACAA CAATTGCATT CATTTTATGT TTCAGGTTCA 9181 GGGGGAGGTG TGGGAGGTTT TTTAAAGCAA GTAAAACCTC TACAAATGTG GTATGGCTGA 9241 TTATGATCCG GCTGCCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG 9301 CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG 9361 GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCGCAGCCA TGAGGTCGAT CGACTCTAGA 9421 GGATCGATCC CCGCCCCGGA CGAACTAAAC CTGACTACGA CATCTCTGCC CCTTCTTCGC 9481 GGGGCAGTGC ATGTAATCCC TTCAGTTGGT TGGTACAACT TGCCAACTGG GCCCTGTTCC 9541 ACATGTGACA CGGGGGGGGA CCAAACACAA AGGGGTTCTC TGACTGTAGT TGACATCCTT 9601 ATAAATGGAT GTGCACATTT GCCAACACTG AGTGGCTTTC ATCCTGGAGC AGACTTTGCA 9661 GTCTGTGGAC TGCAACACAA CATTGCCTTT ATGTGTAACT CTTGGCTGAA GCTCTTACAC 9721 CAATGCTGGG GGACATGTAC CTCCCAGGGG CCCAGGAAGA CTACGGGAGG CTACACCAAC 9781 GTCAATCAGA GGGGCCTGTG TAGCTACCGA TAAGCGGACC CTCAAGAGGG CATTAGCAAT 9841 AGTGTTTATA AGGCCCCCTT GTTAACCCTA AACGGGTAGC ATATGCTTCC CGGGTAGTAG 9901 TATATACTAT CCAGACTAAC CCTAATTCAA TAGCATATGT TACCCAACGG GAAGCATATG 9961 CTATCGAATT AGGGTTAGTA AAAGGGTCCT AAGGAACAGC GATATCTCCC ACCCCATGAG 10021 CTGTCACGGT TTTATTTACA TGGGGTCAGG ATTCCACGAG GGTAGTGAAC CATTTTAGTC 10081 ACAAGGGCAG TGGCTGAAGA TCAAGGAGCG GGCAGTGAAC TCTCCTGAAT CTTCGCCTGC 10141 TTCTTCATTC TCCTTCGTTT AGCTAATAGA ATAACTGCTG AGTTGTGAAC AGTAAGGTGT 10201 ATGTGAGGTG CTCGAAAACA AGGTTTCAGG TGACGCCCCC AGAATAAAAT TTGGACGGGG 10261 GGTTCAGTGG TGGCATTGTG CTATGACACC AATATAACCC TCACAAACCC CTTGGGCAAT 10321 AAATACTAGT GTAGGAATGA AACATTCTGA ATATCTTTAA CAATAGAAAT CCATGGGGTG 10381 GGGACAAGCC GTAAAGACTG GATGTCCATC TCACACGAAT TTATGGCTAT GGGCAACACA 10441 TAATCCTAGT GCAATATGAT ACTGGGGTTA TTAAGATGTG TCCCAGGCAG GGACCAAGAC 10501 AGGTGAACCA TGTTGTTACA CTCTATTTGT AACAAGGGGA AAGAGAGTGG ACGCCGACAG 10561 CAGCGGACTC CACTGGTTGT CTCTAACACC CCCGAAAATT AAACGGGGCT CCACGCCAAT 10621 GGGGCCCATA AACAAAGACA AGTGGCCACT CTTTTTTTTG AAATTGTGGA GTGGGGGCAC 10681 GCGTCAGCCC CCACACGCCG CCCTGCGGTT TTGGACTGTA AAATAAGGGT GTAATAACTT 10741 GGCTGATTGT AACCCCGCTA ACCACTGCGG TCAAACCACT TGCCCACAAA ACCACTAATG 10801 GCACCCCGGG GAATACCTGC ATAAGTAGGT GGGCGGGCCA AGATAGGGGC GCGATTGCTG 10861 CGATCTGGAG GACAAATTAC ACACACTTGC GCCTGAGCGC CAAGCACAGG GTTGTTGGTC 10921 CTCATATTCA CGAGGTCGCT GAGAGCACGG TGGGCTAATG TTGCCATGGG TAGCATATAC 10981 TACCCAAATA TCTGGATAGC ATATGCTATC CTAATCTATA TCTGGGTAGC ATAGGCTATC 11041 CTAATCTATA TCTGGGTAGC ATATGCTATC CTAATCTATA TCTGGGTAGT ATATGCTATC 11101 CTAATTTATA TCTGGGTAGC ATAGGCTATC CTAATCTATA TCTGGGTAGC ATATGCTATC 11161 CTAATCTATA TCTGGGTAGT ATATGCTATC CTAATCTGTA TCCGGGTAGC ATATGCTATC 11221 CTAATAGAGA TTAGGGTAGT ATATGCTATC CTAATTTATA TCTGGGTAGC ATATACTACC 11281 CAAATATCTG GATAGCATAT GCTATCCTAA TCTATATCTG GGTAGCATAT GCTATCCTAA 11341 TCTATATCTG GGTAGCATAG GCTATCCTAA TCTATATCTG GGTAGCATAT GCTATCCTAA 11401 TCTATATCTG GGTAGTATAT GCTATCCTAA TTTATATCTG GGTAGCATAG GCTATCCTAA 11461 TCTATATCTG GGTAGCATAT GCTATCCTAA TCTATATCTG GGTAGTATAT GCTATCCTAA 11521 TCTGTATCCG GGTAGCATAT GCTATCCTCA TGCATATACA GTCAGCATAT GATACCCAGT 11581 AGTAGAGTGG GAGTGCTATC CTTTGCATAT GCCGCCACCT CCCAAGGGGG CGTGAATTTT 11641 CGCTGCTTGT CCTTTTCCTG CATGCTGGTT GCTCCCATTC TTAGGTGAAT TTAAGGAGGC 11701 CAGGCTAAAG CCGTCGCATG TCTGATTGCT CACCAGGTAA ATGTCGCTAA TGTTTTCCAA 11761 CGCGAGAAGG TGTTGAGCGC GGAGCTGAGT GACGTGACAA CATGGGTATG CCCAATTGCC 11821 CCATGTTGGG AGGACGAAAA TGGTGACAAG ACAGATGGCC AGAAATACAC CAACAGCACG 11881 CATGATGTCT ACTGGGGATT TATTCTTTAG TGCGGGGGAA TACACGGCTT TTAATACGAT 11941 TGAGGGCGTC TCCTAACAAG TTACATCACT CCTGCCCTTC CTCACCCTCA TCTCCATCAC 12001 CTCCTTCATC TCCGTCATCT CCGTCATCAC CCTCCGCGGC AGCCCCTTCC ACCATAGGTG 12061 GAAACCAGGG AGGCAAATCT ACTCCATCGT CAAAGCTGCA CACAGTCACC CTGATATTGC 12121 AGGTAGGAGC GGGCTTTGTC ATAACAAGGT CCTTAATCGC ATCCTTCAAA ACCTCAGCAA 12181 ATATATGAGT TTGTAAAAAG ACCATGAAAT AACAGACAAT GGACTCCCTT AGCGGGCCAG 12241 GTTGTGGGCC GGGTCCAGGG GCCATTCCAA AGGGGAGACG ACTCAATGGT GTAAGACGAC 12301 ATTGTGGAAT AGCAAGGGCA GTTCCTCGCC TTAGGTTGTA AAGGGAGGTC TTACTACCTC 12361 CATATACGAA CACACCGGCG ACCCAAGTTC CTTCGTCGGT AGTCCTTTCT ACGTGACTCC 12421 TAGCCAGGAG AGCTCTTAAA CCTTCTGCAA TGTTCTCAAA TTTCGGGTTG GAACCTCCTT 12481 GACCACGATG CTTTCCAAAC CACCCTCCTT TTTTGCGCCT GCCTCCATCA CCCTGACCCC 12541 GGGGTCCAGT GCTTGGGCCT TCTCCTGGGT CATCTGCGGG GCCCTGCTCT ATCGCTCCCG 12601 GGGGCACGTC AGGCTCACCA TCTGGGCCAC CTTCTTGGTG GTATTCAAAA TAATCGGCTT 12661 CCCCTACAGG GTGGAAAAAT GGCCTTCTAC CTGGAGGGGG CCTGCGCGGT GGAGACCCGG 12721 ATGATGATGA CTGACTACTG GGACTCCTGG GCCTCTTTTC TCCACGTCCA CGACCTCTCC 12781 CCCTGGCTCT TTCACGACTT CCCCCCCTGG CTCTTTCACG TCCTCTACCC CGGCGGCCTC 12841 CACTACCTCC TCGACCCCGG CCTCCACTAC CTCCTCGACC CCGGCCTCCA CTGCCTCCTC 12901 GACCCCGGCC TCCACCTCCT GCTCCTGCCC CTCCTGCTCC TGCCCCTCCT CCTGCTCCTG 12961 CCCCTCCTGC CCCTCCTGCT CCTGCCCCTC CTGCCCCTCC TGCTCCTGCC CCTCCTGCCC 13021 CTCCTGCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC CCCTCCTGCC CCTCCTCCTG 13081 CTCCTGCCCC TCCTGCCCCT CCTGCTCCTG CCCCTCCTGC CCCTCCTGCT CCTGCCCCTC 13141 CTGCCCCTCC TGCTCCTGCC CCTCCTGCTC CTGCCCCTCC TGCTCCTGCC CCTCCTGCTC 13201 CTGCCCCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC CCCTCCTGCT CCTGCCCCTC 13261 CTGCCCCTCC TGCCCCTCCT GCTCCTGCCC CTCCTCCTGC TCCTGCCCCT CCTGCCCCTC 13321 CTGCCCCTCC TCCTGCTCCT GCCCCTCCTG CCCCTCCTCC TGCTCCTGCC CCTCCTCCTG 13381 CTCCTGCCCC TCCTGCCCCT CCTGCCCCTC CTCCTGCTCC TGCCCCTCCT GCCCCTCCTC 13441 CTGCTCCTGC CCCTCCTCCT GCTCCTGCCC CTCCTGCCCC TCCTGCCCCT CCTCCTGCTC 13501 CTGCCCCTCC TCCTGCTCCT GCCCCTCCTG CCCCTCCTGC CCCTCCTGCC CCTCCTCCTG 13561 CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC CTGCTCCTGC CCCTCCCGCT CCTGCTCCTG 13621 CTCCTGTTCC ACCGTGGGTC CCTTTGCAGC CAATGCAACT TGGACGTTTT TGGGGTCTCC 13681 GGACACCATC TCTATGTCTT GGCCCTGATC CTGAGCCGCC CGGGGCTCCT GGTCTTCCGC 13741 CTCCTCGTCC TCGTCCTCTT CCCCGTCCTC GTCCATGGTT ATCACCCCCT CTTCTTTGAG 13801 GTCCACTGCC GCCGGAGCCT TCTGGTCCAG ATGTGTCTCC CTTCTCTCCT AGGCCATTTC 13861 CAGGTCCTGT ACCTGGCCCC TCGTCAGACA TGATTCACAC TAAAAGAGAT CAATAGACAT 13921 CTTTATTAGA CGACGCTCAG TGAATACAGG GAGTGCAGAC TCCTGCCCCC TCCAACAGCC 13981 CCCCCACCCT CATCCCCTTC ATGGTCGCTG TCAGACAGAT CCAGGTCTGA AAATTCCCCA 14041 TCCTCCGAAC CATCCTCGTC CTCATCACCA ATTACTCGCA GCCCGGAAAA CTCCCGCTGA 14101 ACATCCTCAA GATTTGCGTC CTGAGCCTCA AGCCAGGCCT CAAATTCCTC GTCCCCCTTT 14161 TTGCTGGACG GTAGGGATGG GGATTCTCGG GACCCCTCCT CTTCCTCTTC AAGGTCACCA 14221 GACAGAGATG CTACTGGGGC AACGGAAGAA AAGCTGGGTG CGGCCTGTGA GGATCAGCTT 14281 ATCGATGATA AGCTGTCAAA CATGAGAATT CTTGAAGACG AAAGGGCCTC GTGATACGCC 14341 TATTTTTATA GGTTAATGTC ATGATAATAA TGGTTTCTTA GACGTCAGGT GGCACTTTTC 14401 GGGGAAATGT GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC 14461 CGCTCATGAG ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTATGA 14521 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT 14581 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG 14641 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG 14701 AACGTTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTG 14761 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG 14821 AGTACTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA 14881 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATCGGAG 14941 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC 15001 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG 15061 CAGCAATGGC AACAACGTTG CGCAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC 15121 GGCAACAATT AATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG 15181 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG 15241 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA 15301 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC 15361 TGATTAAGCA TTGGTAACTG TCAGACCAAG TTTACTCATA TATACTTTAG ATTGATTTAA 15421 AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA 15481 AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG 15541 GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC 15601 CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA 15661 CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC 15721 ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG 15781 TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC 15841 CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC 15901 GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC 15961 CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA 16021 CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC 16081 TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG 16141 CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT 16201 TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCTTTGAG TGAGCTGATA 16261 CCGCTCGCCG CAGCCGAACG ACCGAGCGCA GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC 16321 GCCTGATGCG GTATTTTCTC CTTACGCATC TGTGCGGTAT TTCACACCGC ATATGGTGCA 16381 CTCTCAGTAC AATCTGCTCT GATGCCGCAT AGTTAAGCCA GCTGTGGAAT GTGTGTCAGT 16441 TAGGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA 16501 ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA 16561 GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC 16621 TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG 16681 CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG 16741 GAGGCCTAGG CTTTTGCAAA AAGCTTGCAT GCCTGCAGGT CGGCCGCCAC GACCGGTGCC 16801 GCCACCATCC CCTGACCCAC GCCCCTGACC CCTCACAAGG AGACGACCTT CCATGACCGA 16861 GTACAAGCCC ACGGTGCGCC TCGCCACCCG CGACGACGTC CCCCGGGCCG TACGCACCCT 16921 CGCCGCCGCG TTCGCCGACT ACCCCGCCAC GCGCCACACC GTCGACCCGG ACCGCCACAT 16981 CGAGCGGGTC ACCGAGCTGC AAGAACTCTT CCTCACGCGC GTCGGGCTCG ACATCGGCAA 17041 GGTGTGGGTC GCGGACGACG GCGCCGCGGT GGCGGTCTGG ACCACGCCGG AGAGCGTCGA 17101 AGCGGGGGCG GTGTTCGCCG AGATCGGCCC GCGCATGGCC GAGTTGAGCG GTTCCCGGCT 17161 GGCCGCGCAG CAACAGATGG AAGGCCTCCT GGCGCCGCAC CGGCCCAAGG AGCCCGCGTG 17221 GTTCCTGGCC ACCGTCGGCG TCTCGCCCGA CCACCAGGGC AAGGGTCTGG GCAGCGCCGT 17281 CGTGCTCCCC GGAGTGGAGG CGGCCGAGCG CGCCGGGGTG CCCGCCTTCC TGGAGACCTC 17341 CGCGCCCCGC AACCTCCCCT TCTACGAGCG GCTCGGCTTC ACCGTCACCG CCGACGTCGA 17401 GGTGCCCGAA GGACCGCGCA CCTGGTGCAT GACCCGCAAG CCCGGTGCCT GACGCCCGCC 17461 CCACGACCCG CAGCGCCCGA CCGAAAGGAG CGCACGACCC CATGGCTCCG ACCGAAGCCG 17521 ACCCGGGCGG CCCCGCCGAC CCCGCACCCG CCCCCGAGGC CCACCGACTC TAGAGGATCA 17581 TAATCAGCCA TACCACATTT GTAGAGGTTT TACTTGCTTT AAAAAACCTC CCACACCTCC 17641 CCCTGAACCT GAAACATAAA ATGAATGCAA TTGTTGTTGT TAACTTGTTT ATTGCAGCTT 17701 ATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC 17761 TGCATTCTAG TTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC TGGATCACTC 17821 GCCGATAGTG GAAACCGACG CCCCAGCACT CGTCCGAGGG CAAAGGAATA GGGGAGATGG 17881 GGGAGGCTAA CTGAAACACG GAAGGAGACA ATACCGGAAG GAACCCGCGC TATGACGGCA 17941 ATAAAAAGAC AGAATAAAAC GCACGGGTGT TGGGTCGTTT GTTCATAAAC GCGGGGTTCG 18001 GTCCCAGGGC TGGCACTCTG TCGATACCCC ACCGAGACCC CATTGGGGCC AATACGCCCG 18061 CGTTTCTTCC TTTTCCCCAC CCCACCCCCC AAGTTCGGGT GAAGGCCCAG GGCTCGCAGC 18121 CAACGTCGGG GCGGCAGGCC CTGCCATAGC CACTGGCCCC GTGGGTTAGG GACGGGGTCC 18181 CCCATGGGGA ATGGTTTATG GTTCGTGGGG GTTATTATTT TGGGCGTTGC GTGGGGTCTG 18241 GTCCACGACT GGACTGAGCA GACAGACCCA TGGTTTTTGG ATGGCCTGGG CATGGACCGC 18301 ATGTACTGGC GCGACACGAA CACCGGGCGT CTGTGGCTGC CAAACACCCC CGACCCCCAA 18361 AAACCACCGC GCGGATTTCT GGCGTGCCAA GCTAGTCGAC CAATTCTCAT GTTTGACAGC 18421 TTATCATCGC AGATCCGGGC AACGTTGTTG CATTGCTGCA GGCGCAGAAC TGGTAGGTAT 18481 GGAAGATCTC TAGAAGCTGG GTACCAGCTG CTAGCAAGCT TGCTAGCGGC CGGCTCGAGT 18541 TTACTCCCTA TCAGTGATAG AGAACGTATG TCGAGTTTAC TCCCTATCAG TGATAGAGAA 18601 CGATGTCGAG TTTACTCCCT ATCAGTGATA GAGAACGTAT GTCGAGTTTA CTCCCTATCA 18661 GTGATAGAGA ACGTATGTCG AGTTTACTCC CTATCAGTGA TAGAGAACGT ATGTCGAGTT 18721 TATCCCTATC AGTGATAGAG AACGTATGTC GAGTTTACTC CCTATCAGTG ATAGAGAACG 18781 TATGTCGAGG TAGGCGTGTA CGGTGGGAGG CCTATATAAG CAGAGCTCGT TTAGTGAACC 18841 GTCAGATCGC CG (SEQ ID NO: 39) LINE-1plasmid-CD5_FCR-PI3K_T2A-GFPintron (SEQ ID NO: 40) 1 CGGCCGCGGG GGGAGGAGCC AAGATGGCCG AATAGGAACA GCTCCGGTCT ACAGCTCCCA 61 GCGTGAGCGA CGCAGAAGAC GGTGATTTCT GCATTTCCAT CTGAGGTACC GGGTTCATCT 121 CACTAGGGAG TGCCAGACAG TGGGCGCAGG CCAGTGTGTG TGCGCACCGT GCGCGAGCCG 181 AAGCAGGGCG AGGCATTGCC TCACCTGGGA AGCGCAAGGG GTCAGGGAGT TCCCTTTCCG 241 AGTCAAAGAA AGGGGTGACG GACGCACCTG GAAAATCGGG TCACTCCCAC CCGAATATTG 301 CGCTTTTCAG ACCGGCTTAA GAAACGGCGC ACCACGAGAC TATATCCCAC ACCTGGCTCG 361 GAGGGTCCTA CGCCCACGGA ATCTCGCTGA TTGCTAGCAC AGCAGTCTGA GATCAAACTG 421 CAAGGCGGCA ACGAGGCTGG GGGAGGGGCG CCCGCCATTG CCCAGGCTTG CTTAGGTAAA 481 CAAAGCAGCA GGGAAGCTCG AACTGGGTGG AGCCCACCAC AGCTCAAGGA GGCCTGCCTG 541 CCTCTGTAGG CTCCACCTCT GGGGGCAGGG CACAGACAAA CAAAAAGACA GCAGTAACCT 601 CTGCAGACTT AAGTGTCCCT GTCTGACAGC TTTGAAGAGA GCAGTGGTTC TCCCAGCACG 661 CAGCTGGAGA TCTGAGAACG GGCAGACTGC CTCCTCAAGT GGGTCCCTGA CCCCTGACCC 721 CCGAGCAGCC TAACTGGGAG GCACCCCCCA GCAGGGGCAC ACTGACACCT CACACGGCAG 781 GGTATTCCAA CAGACCTGCA GCTGAGGGTC CTGTCTGTTA GAAGGAAAAC TAACAACCAG 841 AAAGGACATC TACACCGAAA ACCCATCTGT ACATCACCAT CATCAAAGAC CAAAAGTAGA 901 TAAAACCACA AAGATGGGGA AAAAACAGAA CAGAAAAACT GGAAACTCTA AAACGCAGAG 961 CGCCTCTCCT CCTCCAAAGG AACGCAGTTC CTCACCAGCA ACAGAACAAA GCTGGATGGA 1021 GAATGATTTT GATGAGCTGA GAGAAGAAGG CTTCAGACGA TCAAATTACT CTGAGCTACG 1081 GGAGGACATT CAAACCAAAG GCAAAGAAGT TGAAAACTTT GAAAAAAATT TAGAAGAATG 1141 TATAACTAGA ATAACCAATA CAGAGAAGTG CTTAAAGGAG CTGATGGAGC TGAAAACCAA 1201 GGCTCGAGAA CTACGTGAAG AATGCAGAAG CCTCAGGAGC CGATGCGATC AACTGGAAGA 1261 AAGGGTATCA GCAATGGAAG ATGAAATGAA TGAAATGAAG CGAGAAGGGA AGTTTAGAGA 1321 AAAAAGAATA AAAAGAAATG AGCAAAGCCT CCAAGAAATA TGGGACTATG TGAAAAGACC 1381 AAATCTACGT CTGATTGGTG TACCTGAAAG TGATGTGGAG AATGGAACCA AGTTGGAAAA 1441 CACTCTGCAG GATATTATCC AGGAGAACTT CCCCAATCTA GCAAGGCAGG CCAACGTTCA 1501 GATTCAGGAA ATACAGAGAA CGCCACAAAG ATACTCCTCG AGAAGAGCAA CTCCAAGACA 1561 CATAATTGTC AGATTCACCA AAGTTGAAAT GAAGGAAAAA ATGTTAAGGG CAGCCAGAGA 1621 GAAAGGTCGG GTTACCCTCA AAGGAAAGCC CATCAGACTA ACAGCGGATC TCTCGGCAGA 1681 AACCCTACAA GCCAGAAGAG AGTGGGGGCC AATATTCAAC ATTCTTAAAG AAAAGAATTT 1741 TCAACCCAGA ATTTCATATC CAGCCAAACT AAGCTTCATA AGTGAAGGAG AAATAAAATA 1801 CTTTATAGAC AAGCAAATGT TGAGAGATTT TGTCACCACC AGGCCTGCCC TAAAAGAGCT 1861 CCTGAAGGAA GCGCTAAACA TGGAAAGGAA CAACCGGTAC CAGCCGCTGC AAAATCATGC 1921 CAAAATGTAA AGACCATCAA GACTAGGAAG AAACTGCATC AACTAATGAG CAAAATCACC 1981 AGCTAACATC ATAATGACAG GATCAACTTC ACACATAACA ATATTAACTT TAAATATAAA 2041 TGGACTAAAT TCTGCAATTA AAAGACACAG ACTGGCAAGT TGGATAAAGA GTCAAGACCC 2101 ATCAGTGTGC TGTATTCAGG AAACCCATCT CACGTGCAGA GACACACATA GGCTCAAAAT 2161 AAAAGGATGG AGGAAGATCT ACCAAGCCAA TGGAAAACAA AAAAAGGCAG GGGTTGCAAT 2221 CCTAGTCTCT GATAAAACAG ACTTTAAACC AACAAAGATC AAAAGAGACA AAGAAGGCCA 2281 TTACATAATG GTAAAGGGAT CAATTCAACA AGAGGAGCTA ACTATCCTAA ATATTTATGC 2341 ACCCAATACA GGAGCACCCA GATTCATAAA GCAAGTCCTC AGTGACCTAC AAAGAGACTT 2401 AGACTCCCAC ACATTAATAA TGGGAGACTT TAACACCCCA CTGTCAACAT TAGACAGATC 2461 AACGAGACAG AAAGTCAACA AGGATACCCA GGAATTGAAC TCAGCTCTGC ACCAAGCAGA 2521 CCTAATAGAC ATCTACAGAA CTCTCCACCC CAAATCAACA GAATATACAT TTTTTTCAGC 2581 ACCACACCAC ACCTATTCCA AAATTGACCA CATAGTTGGA AGTAAAGCTC TCCTCAGCAA 2641 ATGTAAAAGA ACAGAAATTA TAACAAACTA TCTCTCAGAC CACAGTGCAA TCAAACTAGA 2701 ACTCAGGATT AAGAATCTCA CTCAAAGCCG CTCAACTACA TGGAAACTGA ACAACCTGCT 2761 CCTGAATGAC TACTGGGTAC ATAACGAAAT GAAGGCAGAA ATAAAGATGT TCTTTGAAAC 2821 CAACGAGAAC AAAGACACCA CATACCAGAA TCTCTGGGAC GCATTCAAAG CAGTGTGTAG 2881 AGGGAAATTT ATAGCACTAA ATGCCTACAA GAGAAAGCAG GAAAGATCCA AAATTGACAC 2941 CCTAACATCA CAATTAAAAG AACTAGAAAA GCAAGAGCAA ACACATTCAA AAGCTAGCAG 3001 AAGGCAAGAA ATAACTAAAA TCAGAGCAGA ACTGAAGGAA ATAGAGACAC AAAAAACCCT 3061 TCAAAAAATC AATGAATCCA GGAGCTGGTT TTTTGAAAGG ATCAACAAAA TTGATAGACC 3121 GCTAGCAAGA CTAATAAAGA AAAAAAGAGA GAAGAATCAA ATAGACACAA TAAAAAATGA 3181 TAAAGGGGAT ATCACCACCG ATCCCACAGA AATACAAACT ACCATCAGAG AATACTACAA 3241 ACACCTCTAC GCAAATAAAC TAGAAAATCT AGAAGAAATG GATACATTCC TCGACACATA 3301 CACTCTCCCA AGACTAAACC AGGAAGAAGT TGAATCTCTG AATCGACCAA TAACAGGCTC 3361 TGAAATTGTG GCAATAATCA ATAGTTTACC AACCAAAAAG AGTCCAGGAC CAGATGGATT 3421 CACAGCCGAA TTCTACCAGA GGTACAAGGA GGAACTGGTA CCATTCCTTC TGAAACTATT 3481 CCAATCAATA GAAAAAGAGG GAATCCTCCC TAACTCATTT TATGAGGCCA GCATCATTCT 3541 GATACCAAAG CCGGGCAGAG ACACAACCAA AAAAGAGAAT TTTAGACCAA TATCCTTGAT 3601 GAACATTGAT GCAAAAATCC TCAATAAAAT ACTGGCAAAC CGAATCCAGC AGCACATCAA 3661 AAAGCTTATC CACCATGATC AAGTGGGCTT CATCCCTGGG ATGCAAGGCT GGTTCAATAT 3721 ACGCAAATCA ATAAATGTAA TCCAGCATAT AAACAGAGCC AAAGACAAAA ACCACATGAT 3781 TATCTCAATA GATGCAGAAA AAGCCTTTGA CAAAATTCAA CAACCCTTCA TGCTAAAAAC 3841 TCTCAATAAA TTAGGTATTG ATGGGACGTA TTTCAAAATA ATAAGAGCTA TCTATGACAA 3901 ACCCACAGCC AATATCATAC TGAATGGGCA AAAACTGGAA GCATTCCCTT TGAAAACCGG 3961 CACAAGACAG GGATGCCCTC TCTCACCGCT CCTATTCAAC ATAGTGTTGG AAGTTCTGGC 4021 CAGGGCAATC AGGCAGGAGA AGGAAATAAA GGGTATTCAA TTAGGAAAAG AGGAAGTCAA 4081 ATTGTCCCTG TTTGCAGACG ACATGATTGT TTATCTAGAA AACCCCATCG TCTCAGCCCA 4141 AAATCTCCTT AAGCTGATAA GCAACTTCAG CAAAGTCTCA GGATACAAAA TCAATGTACA 4201 AAAATCACAA GCATTCTTAT ACACCAACAA CAGACAAACA GAGAGCCAAA TCATGGGTGA 4261 ACTCCCATTC ACAATTGCTT CAAAGAGAAT AAAATACCTA GGAATCCAAC TTACAAGGGA 4321 TGTGAAGGAC CTCTTCAAGG AGAACTACAA ACCACTGCTC AAGGAAATAA AAGAGGAGAC 4381 AAACAAATGG AAGAACATTC CATGCTCATG GGTAGGAAGA ATCAATATCG TGAAAATGGC 4441 CATACTGCCC AAGGTAATTT ACAGATTCAA TGCCATCCCC ATCAAGCTAC CAATGACTTT 4501 CTTCACAGAA TTGGAAAAAA CTACTTTAAA GTTCATATGG AACCAAAAAA GAGCCCGCAT 4561 TGCCAAGTCA ATCCTAAGCC AAAAGAACAA AGCTGGAGGC ATCACACTAC CTGACTTCAA 4621 ACTATACTAC AAGGCTACAG TAACCAAAAC AGCATGGTAC TGGTACCAAA ACAGAGATAT 4681 AGATCAATGG AACAGAACAG AGCCCTCAGA AATAATGCCG CATATCTACA ACTATCTGAT 4741 CTTTGACAAA CCTGAGAAAA ACAAGCAATG GGGAAAGGAT TCCCTATTTA ATAAATGGTG 4801 CTGGGAAAAC TGGCTAGCCA TATGTAGAAA GCTGAAACTG GATCCCTTCC TTACACCTTA 4861 TACAAAAATC AATTCAAGAT GGATTAAAGA TTTAAACGTT AAACCTAAAA CCATAAAAAC 4921 CCTAGAAGAA AACCTAGGCA TTACCATTCA GGACATAGGC GTGGGCAAGG ACTTCATGTC 4981 CAAAACACCA AAAGCAATGG CAACAAAAGA CAAAATTGAC AAATGGGATC TAATTAAACT 5041 AAAGAGCTTC TGCACAGCAA AAGAAACTAC CATCAGAGTG AACAGGCAAC CTACAACATG 5101 GGAGAAAATT TTTGCAACCT ACTCATCTGA CAAAGGGCTA ATATCCAGAA TCTACAATGA 5161 ACTCAAACAA ATTTACAAGA AAAAAACAAA CAACCCCATC AAAAAGTGGG CGAAGGACAT 5221 GAACAGACAC TTCTCAAAAG AAGACATTTA TGCAGCCAAA AAACACATGA AGAAATGCTC 5281 ATCATCACTG GCCATCAGAG AAATGCAAAT CAAAACCACT ATGAGATATC ATCTCACACC 5341 AGTTAGAATG GCAATCATTA AAAAGTCAGG AAACAACAGG TGCTGGAGAG GATGCGGAGA 5401 AATAGGAACA CTTTTACACT GTTGGTGGGA CTGTAAACTA GTTCAACCAT TGTGGAAGTC 5461 AGTGTGGCGA TTCCTCAGGG ATCTAGAACT AGAAATACCA TTTGACCCAG CCATCCCATT 5521 ACTGGGTATA TACCCAAATG AGTATAAATC ATGCTGCTAT AAAGACACAT GCACACGTAT 5581 GTTTATTGCG GCACTATTCA CAATAGCAAA GACTTGGAAC CAACCCAAAT GTCCAACAAT 5641 GATAGACTGG ATTAAGAAAA TGTGGCACAT ATACACCATG GAATACTATG CAGCCATAAA 5701 AAATGATGAG TTCATATCCT TTGTAGGGAC ATGGATGAAA TTGGAAACCA TCATTCTCAG 5761 TAAACTATCG CAAGAACAAA AAACCAAACA CCGCATATTC TCACTCATAG GTGGGAATTG 5821 AACAATGAGA TCACATGGAC ACAGGAAGGG GAATATCACA CTCTGGGGAC TGTGGTGGGG 5881 TCGGGGGAGG GGGGAGGGAT AGCATTGGGA GATATACCTA ATGCTAGATG ACACATTAGT 5941 GGGTGCAGCG CACCAGCATG GCACATGTAT ACGGATCCGA ATTCTCGACG GATCGATCCG 6001 AACAAACGAC CCAACACCCG TGCGTTTTAT TCTGTCTTTT TATTGCCGAT CCCCTCAGAA 6061 GAACTCGTCA AGAAGGCGAT AGAAGGCGAT GCGCTGCGAA TCGGGAGCGG CGATACCGTA 6121 AAGCACGAGG AAGCGGTCAG CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC 6181 CAACGCTATG TCCTGATAGC GGTCGGCCGC TTTACTTGTA CAGCTCGTCC ATGCCGAGAG 6241 TGATCCCGGC GGCGGTCACG AACTCCAGCA GGACCATGTG ATCGCGCTTC TCGTTGGGGT 6301 CTTTGCTCAG GGCGGACTGG GTGCTCAGGT AGTGGTTGTC GGGCAGCAGC ACGGGGCCGT 6361 CGCCGATGGG GGTGTTCTGC TGGTAGTGGT CGGCCAGGTG AGTCCAGGAG ATGTTTCAGC 6421 ACTGTTGCCT TTAGTCTCGA GGCAACTTAG ACAACTGAGT ATTGATCTGA GCACAGCAGG 6481 GTGTGAGCTG TTTGAAGATA CTGGGGTTGG GGGTGAAGAA ACTGCAGAGG ACTAACTGGG 6541 CTGAGACCCA GTGGCAATGT TTTAGGGCCT AAGGAATGCC TCTGAAAATC TAGATGGACA 6601 ACTTTGACTT TGAGAAAAGA GAGGTGGAAA TGAGGAAAAT GACTTTTCTT TATTAGATTT 6661 CGGTAGAAAG AACTTTCATC TTTCCCCTAT TTTTGTTATT CGTTTTAAAA CATCTATCTG 6721 GAGGCAGGAC AAGTATGGTC ATTAAAAAGA TGCAGGCAGA AGGCATATAT TGGCTCAGTC 6781 AAAGTGGGGA ACTTTGGTGG CCAAACATAC ATTGCTAAGG CTATTCCTAT ATCAGCTGGA 6841 CACATATAAA ATGCTGCTAA TGCTTCATTA CAAACTTATA TCCTTTAATT CCAGATGGGG 6901 GCAAAGTATG TCCAGGGGTG AGGAACAATT GAAACATTTG GGCTGGAGTA GATTTTGAAA 6961 GTCAGCTCTG TGTGTGTGTG TGTGTGTGTG TGTGTGAGAG CGTGTGTTTC TTTTAACGTT 7021 TTCAGCCTAC AGCATACAGG GTTCATGGTG GCAAGAAGAT AACAAGATTT AAATTATGGC 7081 CAGTGACTAG TGCTGCAAGA AGAACAACTA CCTGCATTTA ATGGGAAAGC AAAATCTCAG 7141 GCTTTGAGGG AAGTTAACAT AGGCTTGATT CTGGGTGGAA GCTGGGTGTG TAGTTATCTG 7201 GAGGCCAGGC TGGAGCTCTC AGCTCACTAT GGGTTCATCT TTATTGTCTC CTTTCATCTC 7261 AACAGCTGCA CGCTGCCGTC CTCGATGTTG TGGCGGATCT TGAAGTTCAC CTTGATGCCG 7321 TTCTTCTGCT TGTCGGCCAT GATATAGACG TTGTGGCTGT TGTAGTTGTA CTCCAGCTTG 7381 TGCCCCAGGA TGTTGCCGTC CTCCTTGAAG TCGATGCCCT TCAGCTCGAT GCGGTTCACC 7441 AGGGTGTCGC CCTCGAACTT CACCTCGGCG CGGGTCTTGT AGTTGCCGTC GTCCTTGAAG 7501 AAGATGGTGC GCTCCTGGAC GTAGCCTTCG GGCATGGCGG ACTTGAAGAA GTCGTGCTGC 7561 TTCATGTGGT CGGGGTAGCG GCTGAAGCAC TGCACGCCGT AGGTCAGGGT GGTCACGAGG 7621 GTGGGCCAGG GCACGGGCAG CTTGCCGGTG GTGCAGATGA ACTTCAGGGT CAGCTTGCCG 7681 TAGGTGGCAT CGCCCTCGCC CTCGCCGGAC ACGCTGAACT TGTGGCCGTT TACGTCGCCG 7741 TCCAGCTCGA CCAGGATGGG CACCACCCCG GTGAACAGCT CCTCGCCCTT GCTCACCATA 7801 GGGCCGGGAT TCTCCTCCAC GTCACCGCAT GTTAGAAGAC TTCCTCTGCC CTCCATGTTC 7861 TCGTAGGAGT CGGCGTCCTC TTCGTGGTTA GGTCCAGGTT GGCCTCTGAT AGACCGCAGC 7921 TGAGGAGCGG CGTACAGAAT GCCTCTCATG TCCTCATAGC TGCCGCTGCC TTGTGGAGGC 7981 TTCTCGTGCT TCAGTGTCTC GTATGTCTCT TGATTCCGGG TGCTCAGGCC GGTGTACACG 8041 CCATCAGATT TCTCGTAGCT GGTGATGGCG GCCTTCCGCA CTTGGATCTT CAGCCGTCTG 8101 CAGTACAGGG TGATGACCAG AGACAGCAGC AGGACACCAC ATGTGCCAGC CAGAGGGGCC 8161 CAAATGTAGA TATCCAGGCC TCTGGTATGC ACAGCTCCGC CTGCAGCAGG TCTACAGGCT 8221 TCAGGTCTGA GAGACAGAGG CTGGCTGGCG ATTGTAGGAG CTGGTGTAGG TGGTCTAGGA 8281 GCGGGTGTTG TTGTAGGCTT GGCGGGCAGA AACACGGGCA CGAAGTGGCT GAAGTACATG 8341 ATGCTATTGC TCAGGGCTCC GCTTCCTCCG CCGCCTGATT TGATTTCCAG CTTGGTGCCT 8401 CCGCCAAATG TCCAAGGGCT CTCGTCGTAC TGCTGGCAGT AGTAGATGCC GAAGTCCTCG 8461 TACTGCAGGC TGCTGATTGT CAGGGTGTAG TCGGTGCCAG AGCCGCTGCC AGAAAATCTG 8521 CTTGGCACGC CGCTTTCCAG TCTGTTGGCC CGGTAGATCA GTGTCTTAGG GGCCTTGCCA 8581 GGCTTCTGCT GGAACCAGCT CAGGTAGCTG TTGATGTCCT GGCTGGCTCT ACAGGTGATG 8641 GTCACTCTAT CGCCCACAGA GGCAGACAGG CTGCTAGGGC TCTGTGTCAT CTGGATATCA 8701 GAGCCACCAC CGCCAGATCC ACCGCCACCT GATCCTCCGC CTCCGCTAGA AACTGTCACT 8761 GTGGTGCCCT GGCCCCACAC ATCGAAGTAC CAGTCGTAGC CTCTTCTGGT GCAGAAGTAC 8821 ACGGCGGTAT CCTCGGCTCT CAGGCTGTTG ATCTGCAGGT AGGCGGTGTT CTTGCTGTCG 8881 TCCAGGCTGA AGGTGAATCT GCCCTTAAAG CTATCGGCGT AGGTTGGCTC GCCGGTGTGG 8941 GTATTGATCC AGCCCATCCA CTCAAGGCCT TTTCCAGGGG CCTGTCGGAC CCAGTTCATG 9001 CCGTAGTTGG TGAAGGTGTA GCCGCTGGCG GCACAGCTGA TTCTGACAGA TCCGCCAGGT 9061 TTCACAAGTC CGCCGCCAGA CTGAACCAGC TGGATCTCAG AGATGCTACA GGCCACTGTT 9121 CCCAGCAGCA GCAGAGACTG CAGCCACATT CGAAGCTTGA GCTCGAGATC TGAGTCCGGT 9181 AGCGCTAGCG GATCTGACGG TTCACTAAAC CAGCTCTGCT TATATAGACC TCCCACCGTA 9241 CACGCCTACC GCCCATTTGC GTCAATGGGG CGGAGTTGTT ACGACATTTT GGAAAGTCCC 9301 GTTGATTTTG GTGCCAAAAC AAACTCCCAT TGACGTCAAT GGGGTGGAGA CTTGGAAATC 9361 CCCGTGAGTC AAACCGCTAT CCACGCCCAT TGATGTACTG CCAAAACCGC ATCACCATGG 9421 TAATAGCGAT GACTAATACG TAGATGTACT GCCAAGTAGG AAAGTCCCAT AAGGTCATGT 9481 ACTGGGCATA ATGCCAGGCG GGCCATTTAC CGTCATTGAC GTCAATAGGG GGCGTACTTG 9541 GCATATGATA CACTTGATGT ACTGCCAAGT GGGCAGTTTA CCGTAAATAC TCCACCCATT 9601 GACGTCAATG GAAAGTCCCT ATTGGCGTTA CTATGGGAAC ATACGTCATT ATTGACGTCA 9661 ATGGGCGGGG GTCGTTGGGC GGTCAGCCAG GCGGGCCATT TACCGTAAGT TATGTAACGC 9721 GGAACTCCAT ATATGGGCTA TGAACTAATG ACCCCGTAAT TGATTACTAT TAGCCCGGGG 9781 GATCCAGACA TGATAAGATA CATTGATGAG TTTGGACAAA CCACAACTAG AATGCAGTGA 9841 AAAAAATGCT TTATTTGTGA AATTTGTGAT GCTATTGCTT TATTTGTAAC CATTATAAGC 9901 TGCAATAAAC AAGTTAACAA CAACAATTGC ATTCATTTTA TGTTTCAGGT TCAGGGGGAG 9961 GTGTGGGAGG TTTTTTAAAG CAAGTAAAAC CTCTACAAAT GTGGTATGGC TGATTATGAT 10021 CCGGCTGCCT CGCGCGTTTC GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG 10081 AGACGGTCAC AGCTTGTCTG TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT 10141 CAGCGGGTGT TGGCGGGTGT CGGGGCGCAG CCATGAGGTC GATCGACTCT AGAGGATCGA 10201 TCCCCGCCCC GGACGAACTA AACCTGACTA CGACATCTCT GCCCCTTCTT CGCGGGGCAG 10261 TGCATGTAAT CCCTTCAGTT GGTTGGTACA ACTTGCCAAC TGGGCCCTGT TCCACATGTG 10321 ACACGGGGGG GGACCAAACA CAAAGGGGTT CTCTGACTGT AGTTGACATC CTTATAAATG 10381 GATGTGCACA TTTGCCAACA CTGAGTGGCT TTCATCCTGG AGCAGACTTT GCAGTCTGTG 10441 GACTGCAACA CAACATTGCC TTTATGTGTA ACTCTTGGCT GAAGCTCTTA CACCAATGCT 10501 GGGGGACATG TACCTCCCAG GGGCCCAGGA AGACTACGGG AGGCTACACC AACGTCAATC 10561 AGAGGGGCCT GTGTAGCTAC CGATAAGCGG ACCCTCAAGA GGGCATTAGC AATAGTGTTT 10621 ATAAGGCCCC CTTGTTAACC CTAAACGGGT AGCATATGCT TCCCGGGTAG TAGTATATAC 10681 TATCCAGACT AACCCTAATT CAATAGCATA TGTTACCCAA CGGGAAGCAT ATGCTATCGA 10741 ATTAGGGTTA GTAAAAGGGT CCTAAGGAAC AGCGATATCT CCCACCCCAT GAGCTGTCAC 10801 GGTTTTATTT ACATGGGGTC AGGATTCCAC GAGGGTAGTG AACCATTTTA GTCACAAGGG 10861 CAGTGGCTGA AGATCAAGGA GCGGGCAGTG AACTCTCCTG AATCTTCGCC TGCTTCTTCA 10921 TTCTCCTTCG TTTAGCTAAT AGAATAACTG CTGAGTTGTG AACAGTAAGG TGTATGTGAG 10981 GTGCTCGAAA ACAAGGTTTC AGGTGACGCC CCCAGAATAA AATTTGGACG GGGGGTTCAG 11041 TGGTGGCATT GTGCTATGAC ACCAATATAA CCCTCACAAA CCCCTTGGGC AATAAATACT 11101 AGTGTAGGAA TGAAACATTC TGAATATCTT TAACAATAGA AATCCATGGG GTGGGGACAA 11161 GCCGTAAAGA CTGGATGTCC ATCTCACACG AATTTATGGC TATGGGCAAC ACATAATCCT 11221 AGTGCAATAT GATACTGGGG TTATTAAGAT GTGTCCCAGG CAGGGACCAA GACAGGTGAA 11281 CCATGTTGTT ACACTCTATT TGTAACAAGG GGAAAGAGAG TGGACGCCGA CAGCAGCGGA 11341 CTCCACTGGT TGTCTCTAAC ACCCCCGAAA ATTAAACGGG GCTCCACGCC AATGGGGCCC 11401 ATAAACAAAG ACAAGTGGCC ACTCTTTTTT TTGAAATTGT GGAGTGGGGG CACGCGTCAG 11461 CCCCCACACG CCGCCCTGCG GTTTTGGACT GTAAAATAAG GGTGTAATAA CTTGGCTGAT 11521 TGTAACCCCG CTAACCACTG CGGTCAAACC ACTTGCCCAC AAAACCACTA ATGGCACCCC 11581 GGGGAATACC TGCATAAGTA GGTGGGCGGG CCAAGATAGG GGCGCGATTG CTGCGATCTG 11641 GAGGACAAAT TACACACACT TGCGCCTGAG CGCCAAGCAC AGGGTTGTTG GTCCTCATAT 11701 TCACGAGGTC GCTGAGAGCA CGGTGGGCTA ATGTTGCCAT GGGTAGCATA TACTACCCAA 11761 ATATCTGGAT AGCATATGCT ATCCTAATCT ATATCTGGGT AGCATAGGCT ATCCTAATCT 11821 ATATCTGGGT AGCATATGCT ATCCTAATCT ATATCTGGGT AGTATATGCT ATCCTAATTT 11881 ATATCTGGGT AGCATAGGCT ATCCTAATCT ATATCTGGGT AGCATATGCT ATCCTAATCT 11941 ATATCTGGGT AGTATATGCT ATCCTAATCT GTATCCGGGT AGCATATGCT ATCCTAATAG 12001 AGATTAGGGT AGTATATGCT ATCCTAATTT ATATCTGGGT AGCATATACT ACCCAAATAT 12061 CTGGATAGCA TATGCTATCC TAATCTATAT CTGGGTAGCA TATGCTATCC TAATCTATAT 12121 CTGGGTAGCA TAGGCTATCC TAATCTATAT CTGGGTAGCA TATGCTATCC TAATCTATAT 12181 CTGGGTAGTA TATGCTATCC TAATTTATAT CTGGGTAGCA TAGGCTATCC TAATCTATAT 12241 CTGGGTAGCA TATGCTATCC TAATCTATAT CTGGGTAGTA TATGCTATCC TAATCTGTAT 12301 CCGGGTAGCA TATGCTATCC TCATGCATAT ACAGTCAGCA TATGATACCC AGTAGTAGAG 12361 TGGGAGTGCT ATCCTTTGCA TATGCCGCCA CCTCCCAAGG GGGCGTGAAT TTTCGCTGCT 12421 TGTCCTTTTC CTGCATGCTG GTTGCTCCCA TTCTTAGGTG AATTTAAGGA GGCCAGGCTA 12481 AAGCCGTCGC ATGTCTGATT GCTCACCAGG TAAATGTCGC TAATGTTTTC CAACGCGAGA 12541 AGGTGTTGAG CGCGGAGCTG AGTGACGTGA CAACATGGGT ATGCCCAATT GCCCCATGTT 12601 GGGAGGACGA AAATGGTGAC AAGACAGATG GCCAGAAATA CACCAACAGC ACGCATGATG 12661 TCTACTGGGG ATTTATTCTT TAGTGCGGGG GAATACACGG CTTTTAATAC GATTGAGGGC 12721 GTCTCCTAAC AAGTTACATC ACTCCTGCCC TTCCTCACCC TCATCTCCAT CACCTCCTTC 12781 ATCTCCGTCA TCTCCGTCAT CACCCTCCGC GGCAGCCCCT TCCACCATAG GTGGAAACCA 12841 GGGAGGCAAA TCTACTCCAT CGTCAAAGCT GCACACAGTC ACCCTGATAT TGCAGGTAGG 12901 AGCGGGCTTT GTCATAACAA GGTCCTTAAT CGCATCCTTC AAAACCTCAG CAAATATATG 12961 AGTTTGTAAA AAGACCATGA AATAACAGAC AATGGACTCC CTTAGCGGGC CAGGTTGTGG 13021 GCCGGGTCCA GGGGCCATTC CAAAGGGGAG ACGACTCAAT GGTGTAAGAC GACATTGTGG 13081 AATAGCAAGG GCAGTTCCTC GCCTTAGGTT GTAAAGGGAG GTCTTACTAC CTCCATATAC 13141 GAACACACCG GCGACCCAAG TTCCTTCGTC GGTAGTCCTT TCTACGTGAC TCCTAGCCAG 13201 GAGAGCTCTT AAACCTTCTG CAATGTTCTC AAATTTCGGG TTGGAACCTC CTTGACCACG 13261 ATGCTTTCCA AACCACCCTC CTTTTTTGCG CCTGCCTCCA TCACCCTGAC CCCGGGGTCC 13321 AGTGCTTGGG CCTTCTCCTG GGTCATCTGC GGGGCCCTGC TCTATCGCTC CCGGGGGCAC 13381 GTCAGGCTCA CCATCTGGGC CACCTTCTTG GTGGTATTCA AAATAATCGG CTTCCCCTAC 13441 AGGGTGGAAA AATGGCCTTC TACCTGGAGG GGGCCTGCGC GGTGGAGACC CGGATGATGA 13501 TGACTGACTA CTGGGACTCC TGGGCCTCTT TTCTCCACGT CCACGACCTC TCCCCCTGGC 13561 TCTTTCACGA CTTCCCCCCC TGGCTCTTTC ACGTCCTCTA CCCCGGCGGC CTCCACTACC 13621 TCCTCGACCC CGGCCTCCAC TACCTCCTCG ACCCCGGCCT CCACTGCCTC CTCGACCCCG 13681 GCCTCCACCT CCTGCTCCTG CCCCTCCTGC TCCTGCCCCT CCTCCTGCTC CTGCCCCTCC 13741 TGCCCCTCCT GCTCCTGCCC CTCCTGCCCC TCCTGCTCCT GCCCCTCCTG CCCCTCCTGC 13801 TCCTGCCCCT CCTGCCCCTC CTCCTGCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC 13861 CCCTCCTGCC CCTCCTGCTC CTGCCCCTCC TGCCCCTCCT GCTCCTGCCC CTCCTGCCCC 13921 TCCTGCTCCT GCCCCTCCTG CTCCTGCCCC TCCTGCTCCT GCCCCTCCTG CTCCTGCCCC 13981 TCCTGCCCCT CCTGCCCCTC CTCCTGCTCC TGCCCCTCCT GCTCCTGCCC CTCCTGCCCC 14041 TCCTGCCCCT CCTGCTCCTG CCCCTCCTCC TGCTCCTGCC CCTCCTGCCC CTCCTGCCCC 14101 TCCTCCTGCT CCTGCCCCTC CTGCCCCTCC TCCTGCTCCT GCCCCTCCTC CTGCTCCTGC 14161 CCCTCCTGCC CCTCCTGCCC CTCCTCCTGC TCCTGCCCCT CCTGCCCCTC CTCCTGCTCC 14221 TGCCCCTCCT CCTGCTCCTG CCCCTCCTGC CCCTCCTGCC CCTCCTCCTG CTCCTGCCCC 14281 TCCTCCTGCT CCTGCCCCTC CTGCCCCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC 14341 CCCTCCTCCT GCTCCTGCCC CTCCTGCTCC TGCCCCTCCC GCTCCTGCTC CTGCTCCTGT 14401 TCCACCGTGG GTCCCTTTGC AGCCAATGCA ACTTGGACGT TTTTGGGGTC TCCGGACACC 14461 ATCTCTATGT CTTGGCCCTG ATCCTGAGCC GCCCGGGGCT CCTGGTCTTC CGCCTCCTCG 14521 TCCTCGTCCT CTTCCCCGTC CTCGTCCATG GTTATCACCC CCTCTTCTTT GAGGTCCACT 14581 GCCGCCGGAG CCTTCTGGTC CAGATGTGTC TCCCTTCTCT CCTAGGCCAT TTCCAGGTCC 14641 TGTACCTGGC CCCTCGTCAG ACATGATTCA CACTAAAAGA GATCAATAGA CATCTTTATT 14701 AGACGACGCT CAGTGAATAC AGGGAGTGCA GACTCCTGCC CCCTCCAACA GCCCCCCCAC 14761 CCTCATCCCC TTCATGGTCG CTGTCAGACA GATCCAGGTC TGAAAATTCC CCATCCTCCG 14821 AACCATCCTC GTCCTCATCA CCAATTACTC GCAGCCCGGA AAACTCCCGC TGAACATCCT 14881 CAAGATTTGC GTCCTGAGCC TCAAGCCAGG CCTCAAATTC CTCGTCCCCC TTTTTGCTGG 14941 ACGGTAGGGA TGGGGATTCT CGGGACCCCT CCTCTTCCTC TTCAAGGTCA CCAGACAGAG 15001 ATGCTACTGG GGCAACGGAA GAAAAGCTGG GTGCGGCCTG TGAGGATCAG CTTATCGATG 15061 ATAAGCTGTC AAACATGAGA ATTCTTGAAG ACGAAAGGGC CTCGTGATAC GCCTATTTTT 15121 ATAGGTTAAT GTCATGATAA TAATGGTTTC TTAGACGTCA GGTGGCACTT TTCGGGGAAA 15181 TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT 15241 GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA TGAGTATTCA 15301 ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG TTTTTGCTCA 15361 CCCAGAAACG CTGGTGAAAG TAAAAGATGC TGAAGATCAG TTGGGTGCAC GAGTGGGTTA 15421 CATCGAACTG GATCTCAACA GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG AAGAACGTTT 15481 TCCAATGATG AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTGTTGACGC 15541 CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG TTGAGTACTC 15601 ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT GCAGTGCTGC 15661 CATAACCATG AGTGATAACA CTGCGGCCAA CTTACTTCTG ACAACGATCG GAGGACCGAA 15721 GGAGCTAACC GCTTTTTTGC ACAACATGGG GGATCATGTA ACTCGCCTTG ATCGTTGGGA 15781 ACCGGAGCTG AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC CTGCAGCAAT 15841 GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT CCCGGCAACA 15901 ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT CGGCCCTTCC 15961 GGCTGGCTGG TTTATTGCTG ATAAATCTGG AGCCGGTGAG CGTGGGTCTC GCGGTATCAT 16021 TGCAGCACTG GGGCCAGATG GTAAGCCCTC CCGTATCGTA GTTATCTACA CGACGGGGAG 16081 TCAGGCAACT ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA 16141 GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT TAAAACTTCA 16201 TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC 16261 TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC 16321 TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC 16381 AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT 16441 CAGCAGAGCG CAGATACCAA ATACTGTCCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT 16501 CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC 16561 TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA 16621 GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC 16681 CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG 16741 GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA 16801 GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT 16861 TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA 16921 CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC 16981 GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCTTT GAGTGAGCTG ATACCGCTCG 17041 CCGCAGCCGA ACGACCGAGC GCAGCGAGTC AGTGAGCGAG GAAGCGGAAG AGCGCCTGAT 17101 GCGGTATTTT CTCCTTACGC ATCTGTGCGG TATTTCACAC CGCATATGGT GCACTCTCAG 17161 TACAATCTGC TCTGATGCCG CATAGTTAAG CCAGCTGTGG AATGTGTGTC AGTTAGGGTG 17221 TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC 17281 AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA 17341 TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC 17401 GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC 17461 CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT 17521 AGGCTTTTGC AAAAAGCTTG CATGCCTGCA GGTCGGCCGC CACGACCGGT GCCGCCACCA 17581 TCCCCTGACC CACGCCCCTG ACCCCTCACA AGGAGACGAC CTTCCATGAC CGAGTACAAG 17641 CCCACGGTGC GCCTCGCCAC CCGCGACGAC GTCCCCCGGG CCGTACGCAC CCTCGCCGCC 17701 GCGTTCGCCG ACTACCCCGC CACGCGCCAC ACCGTCGACC CGGACCGCCA CATCGAGCGG 17761 GTCACCGAGC TGCAAGAACT CTTCCTCACG CGCGTCGGGC TCGACATCGG CAAGGTGTGG 17821 GTCGCGGACG ACGGCGCCGC GGTGGCGGTC TGGACCACGC CGGAGAGCGT CGAAGCGGGG 17881 GCGGTGTTCG CCGAGATCGG CCCGCGCATG GCCGAGTTGA GCGGTTCCCG GCTGGCCGCG 17941 CAGCAACAGA TGGAAGGCCT CCTGGCGCCG CACCGGCCCA AGGAGCCCGC GTGGTTCCTG 18001 GCCACCGTCG GCGTCTCGCC CGACCACCAG GGCAAGGGTC TGGGCAGCGC CGTCGTGCTC 18061 CCCGGAGTGG AGGCGGCCGA GCGCGCCGGG GTGCCCGCCT TCCTGGAGAC CTCCGCGCCC 18121 CGCAACCTCC CCTTCTACGA GCGGCTCGGC TTCACCGTCA CCGCCGACGT CGAGGTGCCC 18181 GAAGGACCGC GCACCTGGTG CATGACCCGC AAGCCCGGTG CCTGACGCCC GCCCCACGAC 18241 CCGCAGCGCC CGACCGAAAG GAGCGCACGA CCCCATGGCT CCGACCGAAG CCGACCCGGG 18301 CGGCCCCGCC GACCCCGCAC CCGCCCCCGA GGCCCACCGA CTCTAGAGGA TCATAATCAG 18361 CCATACCACA TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA 18421 CCTGAAACAT AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG 18481 TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC 18541 TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGGATCA CTCGCCGATA 18601 GTGGAAACCG ACGCCCCAGC ACTCGTCCGA GGGCAAAGGA ATAGGGGAGA TGGGGGAGGC 18661 TAACTGAAAC ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACG GCAATAAAAA 18721 GACAGAATAA AACGCACGGG TGTTGGGTCG TTTGTTCATA AACGCGGGGT TCGGTCCCAG 18781 GGCTGGCACT CTGTCGATAC CCCACCGAGA CCCCATTGGG GCCAATACGC CCGCGTTTCT 18841 TCCTTTTCCC CACCCCACCC CCCAAGTTCG GGTGAAGGCC CAGGGCTCGC AGCCAACGTC 18901 GGGGCGGCAG GCCCTGCCAT AGCCACTGGC CCCGTGGGTT AGGGACGGGG TCCCCCATGG 18961 GGAATGGTTT ATGGTTCGTG GGGGTTATTA TTTTGGGCGT TGCGTGGGGT CTGGTCCACG 19021 ACTGGACTGA GCAGACAGAC CCATGGTTTT TGGATGGCCT GGGCATGGAC CGCATGTACT 19081 GGCGCGACAC GAACACCGGG CGTCTGTGGC TGCCAAACAC CCCCGACCCC CAAAAACCAC 19141 CGCGCGGATT TCTGGCGTGC CAAGCTAGTC GACCAATTCT CATGTTTGAC AGCTTATCAT 19201 CGCAGATCCG GGCAACGTTG TTGCATTGCT GCAGGCGCAG AACTGGTAGG TATGGAAGAT 19261 CTCTAGAAGC TGGGTACCAG CTGCTAGCAA GCTTGCTAGC GGCCGGCTCG AGTTTACTCC 19321 CTATCAGTGA TAGAGAACGT ATGTCGAGTT TACTCCCTAT CAGTGATAGA GAACGATGTC 19381 GAGTTTACTC CCTATCAGTG ATAGAGAACG TATGTCGAGT TTACTCCCTA TCAGTGATAG 19441 AGAACGTATG TCGAGTTTAC TCCCTATCAG TGATAGAGAA CGTATGTCGA GTTTATCCCT 19501 ATCAGTGATA GAGAACGTAT GTCGAGTTTA CTCCCTATCA GTGATAGAGA ACGTATGTCG 19561 AGGTAGGCGT GTACGGTGGG AGGCCTATAT AAGCAGAGCT CGTTTAGTGA ACCGTCAGAT 19621 CGCCG (SEQ ID NO: 40) LINE-1 plasmid Her2-Cd3z-T2A GFPintron (SEQ ID NO: 41) 1 CGGCCGCGGG GGGAGGAGCC AAGATGGCCG AATAGGAACA GCTCCGGTCT ACAGCTCCCA 61 GCGTGAGCGA CGCAGAAGAC GGTGATTTCT GCATTTCCAT CTGAGGTACC GGGTTCATCT 121 CACTAGGGAG TGCCAGACAG TGGGCGCAGG CCAGTGTGTG TGCGCACCGT GCGCGAGCCG 181 AAGCAGGGCG AGGCATTGCC TCACCTGGGA AGCGCAAGGG GTCAGGGAGT TCCCTTTCCG 241 AGTCAAAGAA AGGGGTGACG GACGCACCTG GAAAATCGGG TCACTCCCAC CCGAATATTG 301 CGCTTTTCAG ACCGGCTTAA GAAACGGCGC ACCACGAGAC TATATCCCAC ACCTGGCTCG 361 GAGGGTCCTA CGCCCACGGA ATCTCGCTGA TTGCTAGCAC AGCAGTCTGA GATCAAACTG 421 CAAGGCGGCA ACGAGGCTGG GGGAGGGGCG CCCGCCATTG CCCAGGCTTG CTTAGGTAAA 481 CAAAGCAGCA GGGAAGCTCG AACTGGGTGG AGCCCACCAC AGCTCAAGGA GGCCTGCCTG 541 CCTCTGTAGG CTCCACCTCT GGGGGCAGGG CACAGACAAA CAAAAAGACA GCAGTAACCT 601 CTGCAGACTT AAGTGTCCCT GTCTGACAGC TTTGAAGAGA GCAGTGGTTC TCCCAGCACG 661 CAGCTGGAGA TCTGAGAACG GGCAGACTGC CTCCTCAAGT GGGTCCCTGA CCCCTGACCC 721 CCGAGCAGCC TAACTGGGAG GCACCCCCCA GCAGGGGCAC ACTGACACCT CACACGGCAG 781 GGTATTCCAA CAGACCTGCA GCTGAGGGTC CTGTCTGTTA GAAGGAAAAC TAACAACCAG 841 AAAGGACATC TACACCGAAA ACCCATCTGT ACATCACCAT CATCAAAGAC CAAAAGTAGA 901 TAAAACCACA AAGATGGGGA AAAAACAGAA CAGAAAAACT GGAAACTCTA AAACGCAGAG 961 CGCCTCTCCT CCTCCAAAGG AACGCAGTTC CTCACCAGCA ACAGAACAAA GCTGGATGGA 1021 GAATGATTTT GATGAGCTGA GAGAAGAAGG CTTCAGACGA TCAAATTACT CTGAGCTACG 1081 GGAGGACATT CAAACCAAAG GCAAAGAAGT TGAAAACTTT GAAAAAAATT TAGAAGAATG 1141 TATAACTAGA ATAACCAATA CAGAGAAGTG CTTAAAGGAG CTGATGGAGC TGAAAACCAA 1201 GGCTCGAGAA CTACGTGAAG AATGCAGAAG CCTCAGGAGC CGATGCGATC AACTGGAAGA 1261 AAGGGTATCA GCAATGGAAG ATGAAATGAA TGAAATGAAG CGAGAAGGGA AGTTTAGAGA 1321 AAAAAGAATA AAAAGAAATG AGCAAAGCCT CCAAGAAATA TGGGACTATG TGAAAAGACC 1381 AAATCTACGT CTGATTGGTG TACCTGAAAG TGATGTGGAG AATGGAACCA AGTTGGAAAA 1441 CACTCTGCAG GATATTATCC AGGAGAACTT CCCCAATCTA GCAAGGCAGG CCAACGTTCA 1501 GATTCAGGAA ATACAGAGAA CGCCACAAAG ATACTCCTCG AGAAGAGCAA CTCCAAGACA 1561 CATAATTGTC AGATTCACCA AAGTTGAAAT GAAGGAAAAA ATGTTAAGGG CAGCCAGAGA 1621 GAAAGGTCGG GTTACCCTCA AAGGAAAGCC CATCAGACTA ACAGCGGATC TCTCGGCAGA 1681 AACCCTACAA GCCAGAAGAG AGTGGGGGCC AATATTCAAC ATTCTTAAAG AAAAGAATTT 1741 TCAACCCAGA ATTTCATATC CAGCCAAACT AAGCTTCATA AGTGAAGGAG AAATAAAATA 1801 CTTTATAGAC AAGCAAATGT TGAGAGATTT TGTCACCACC AGGCCTGCCC TAAAAGAGCT 1861 CCTGAAGGAA GCGCTAAACA TGGAAAGGAA CAACCGGTAC CAGCCGCTGC AAAATCATGC 1921 CAAAATGTAA AGACCATCAA GACTAGGAAG AAACTGCATC AACTAATGAG CAAAATCACC 1981 AGCTAACATC ATAATGACAG GATCAACTTC ACACATAACA ATATTAACTT TAAATATAAA 2041 TGGACTAAAT TCTGCAATTA AAAGACACAG ACTGGCAAGT TGGATAAAGA GTCAAGACCC 2101 ATCAGTGTGC TGTATTCAGG AAACCCATCT CACGTGCAGA GACACACATA GGCTCAAAAT 2161 AAAAGGATGG AGGAAGATCT ACCAAGCCAA TGGAAAACAA AAAAAGGCAG GGGTTGCAAT 2221 CCTAGTCTCT GATAAAACAG ACTTTAAACC AACAAAGATC AAAAGAGACA AAGAAGGCCA 2281 TTACATAATG GTAAAGGGAT CAATTCAACA AGAGGAGCTA ACTATCCTAA ATATTTATGC 2341 ACCCAATACA GGAGCACCCA GATTCATAAA GCAAGTCCTC AGTGACCTAC AAAGAGACTT 2401 AGACTCCCAC ACATTAATAA TGGGAGACTT TAACACCCCA CTGTCAACAT TAGACAGATC 2461 AACGAGACAG AAAGTCAACA AGGATACCCA GGAATTGAAC TCAGCTCTGC ACCAAGCAGA 2521 CCTAATAGAC ATCTACAGAA CTCTCCACCC CAAATCAACA GAATATACAT TTTTTTCAGC 2581 ACCACACCAC ACCTATTCCA AAATTGACCA CATAGTTGGA AGTAAAGCTC TCCTCAGCAA 2641 ATGTAAAAGA ACAGAAATTA TAACAAACTA TCTCTCAGAC CACAGTGCAA TCAAACTAGA 2701 ACTCAGGATT AAGAATCTCA CTCAAAGCCG CTCAACTACA TGGAAACTGA ACAACCTGCT 2761 CCTGAATGAC TACTGGGTAC ATAACGAAAT GAAGGCAGAA ATAAAGATGT TCTTTGAAAC 2821 CAACGAGAAC AAAGACACCA CATACCAGAA TCTCTGGGAC GCATTCAAAG CAGTGTGTAG 2881 AGGGAAATTT ATAGCACTAA ATGCCTACAA GAGAAAGCAG GAAAGATCCA AAATTGACAC 2941 CCTAACATCA CAATTAAAAG AACTAGAAAA GCAAGAGCAA ACACATTCAA AAGCTAGCAG 3001 AAGGCAAGAA ATAACTAAAA TCAGAGCAGA ACTGAAGGAA ATAGAGACAC AAAAAACCCT 3061 TCAAAAAATC AATGAATCCA GGAGCTGGTT TTTTGAAAGG ATCAACAAAA TTGATAGACC 3121 GCTAGCAAGA CTAATAAAGA AAAAAAGAGA GAAGAATCAA ATAGACACAA TAAAAAATGA 3181 TAAAGGGGAT ATCACCACCG ATCCCACAGA AATACAAACT ACCATCAGAG AATACTACAA 3241 ACACCTCTAC GCAAATAAAC TAGAAAATCT AGAAGAAATG GATACATTCC TCGACACATA 3301 CACTCTCCCA AGACTAAACC AGGAAGAAGT TGAATCTCTG AATCGACCAA TAACAGGCTC 3361 TGAAATTGTG GCAATAATCA ATAGTTTACC AACCAAAAAG AGTCCAGGAC CAGATGGATT 3421 CACAGCCGAA TTCTACCAGA GGTACAAGGA GGAACTGGTA CCATTCCTTC TGAAACTATT 3481 CCAATCAATA GAAAAAGAGG GAATCCTCCC TAACTCATTT TATGAGGCCA GCATCATTCT 3541 GATACCAAAG CCGGGCAGAG ACACAACCAA AAAAGAGAAT TTTAGACCAA TATCCTTGAT 3601 GAACATTGAT GCAAAAATCC TCAATAAAAT ACTGGCAAAC CGAATCCAGC AGCACATCAA 3661 AAAGCTTATC CACCATGATC AAGTGGGCTT CATCCCTGGG ATGCAAGGCT GGTTCAATAT 3721 ACGCAAATCA ATAAATGTAA TCCAGCATAT AAACAGAGCC AAAGACAAAA ACCACATGAT 3781 TATCTCAATA GATGCAGAAA AAGCCTTTGA CAAAATTCAA CAACCCTTCA TGCTAAAAAC 3841 TCTCAATAAA TTAGGTATTG ATGGGACGTA TTTCAAAATA ATAAGAGCTA TCTATGACAA 3901 ACCCACAGCC AATATCATAC TGAATGGGCA AAAACTGGAA GCATTCCCTT TGAAAACCGG 3961 CACAAGACAG GGATGCCCTC TCTCACCGCT CCTATTCAAC ATAGTGTTGG AAGTTCTGGC 4021 CAGGGCAATC AGGCAGGAGA AGGAAATAAA GGGTATTCAA TTAGGAAAAG AGGAAGTCAA 4081 ATTGTCCCTG TTTGCAGACG ACATGATTGT TTATCTAGAA AACCCCATCG TCTCAGCCCA 4141 AAATCTCCTT AAGCTGATAA GCAACTTCAG CAAAGTCTCA GGATACAAAA TCAATGTACA 4201 AAAATCACAA GCATTCTTAT ACACCAACAA CAGACAAACA GAGAGCCAAA TCATGGGTGA 4261 ACTCCCATTC ACAATTGCTT CAAAGAGAAT AAAATACCTA GGAATCCAAC TTACAAGGGA 4321 TGTGAAGGAC CTCTTCAAGG AGAACTACAA ACCACTGCTC AAGGAAATAA AAGAGGAGAC 4381 AAACAAATGG AAGAACATTC CATGCTCATG GGTAGGAAGA ATCAATATCG TGAAAATGGC 4441 CATACTGCCC AAGGTAATTT ACAGATTCAA TGCCATCCCC ATCAAGCTAC CAATGACTTT 4501 CTTCACAGAA TTGGAAAAAA CTACTTTAAA GTTCATATGG AACCAAAAAA GAGCCCGCAT 4561 TGCCAAGTCA ATCCTAAGCC AAAAGAACAA AGCTGGAGGC ATCACACTAC CTGACTTCAA 4621 ACTATACTAC AAGGCTACAG TAACCAAAAC AGCATGGTAC TGGTACCAAA ACAGAGATAT 4681 AGATCAATGG AACAGAACAG AGCCCTCAGA AATAATGCCG CATATCTACA ACTATCTGAT 4741 CTTTGACAAA CCTGAGAAAA ACAAGCAATG GGGAAAGGAT TCCCTATTTA ATAAATGGTG 4801 CTGGGAAAAC TGGCTAGCCA TATGTAGAAA GCTGAAACTG GATCCCTTCC TTACACCTTA 4861 TACAAAAATC AATTCAAGAT GGATTAAAGA TTTAAACGTT AAACCTAAAA CCATAAAAAC 4921 CCTAGAAGAA AACCTAGGCA TTACCATTCA GGACATAGGC GTGGGCAAGG ACTTCATGTC 4981 CAAAACACCA AAAGCAATGG CAACAAAAGA CAAAATTGAC AAATGGGATC TAATTAAACT 5041 AAAGAGCTTC TGCACAGCAA AAGAAACTAC CATCAGAGTG AACAGGCAAC CTACAACATG 5101 GGAGAAAATT TTTGCAACCT ACTCATCTGA CAAAGGGCTA ATATCCAGAA TCTACAATGA 5161 ACTCAAACAA ATTTACAAGA AAAAAACAAA CAACCCCATC AAAAAGTGGG CGAAGGACAT 5221 GAACAGACAC TTCTCAAAAG AAGACATTTA TGCAGCCAAA AAACACATGA AGAAATGCTC 5281 ATCATCACTG GCCATCAGAG AAATGCAAAT CAAAACCACT ATGAGATATC ATCTCACACC 5341 AGTTAGAATG GCAATCATTA AAAAGTCAGG AAACAACAGG TGCTGGAGAG GATGCGGAGA 5401 AATAGGAACA CTTTTACACT GTTGGTGGGA CTGTAAACTA GTTCAACCAT TGTGGAAGTC 5461 AGTGTGGCGA TTCCTCAGGG ATCTAGAACT AGAAATACCA TTTGACCCAG CCATCCCATT 5521 ACTGGGTATA TACCCAAATG AGTATAAATC ATGCTGCTAT AAAGACACAT GCACACGTAT 5581 GTTTATTGCG GCACTATTCA CAATAGCAAA GACTTGGAAC CAACCCAAAT GTCCAACAAT 5641 GATAGACTGG ATTAAGAAAA TGTGGCACAT ATACACCATG GAATACTATG CAGCCATAAA 5701 AAATGATGAG TTCATATCCT TTGTAGGGAC ATGGATGAAA TTGGAAACCA TCATTCTCAG 5761 TAAACTATCG CAAGAACAAA AAACCAAACA CCGCATATTC TCACTCATAG GTGGGAATTG 5821 AACAATGAGA TCACATGGAC ACAGGAAGGG GAATATCACA CTCTGGGGAC TGTGGTGGGG 5881 TCGGGGGAGG GGGGAGGGAT AGCATTGGGA GATATACCTA ATGCTAGATG ACACATTAGT 5941 GGGTGCAGCG CACCAGCATG GCACATGTAT ACGGATCCGA ATTCTCGACG GATCGATCCG 6001 AACAAACGAC CCAACACCCG TGCGTTTTAT TCTGTCTTTT TATTGCCGAT CCCCTCAGAA 6061 GAACTCGTCA AGAAGGCGAT AGAAGGCGAT GCGCTGCGAA TCGGGAGCGG CGATACCGTA 6121 AAGCACGAGG AAGCGGTCAG CCCATTCGCC GCCAAGCTCT TCAGCAATAT CACGGGTAGC 6181 CAACGCTATG TCCTGATAGC GGTCGGCCGC TTTACTTGTA CAGCTCGTCC ATGCCGAGAG 6241 TGATCCCGGC GGCGGTCACG AACTCCAGCA GGACCATGTG ATCGCGCTTC TCGTTGGGGT 6301 CTTTGCTCAG GGCGGACTGG GTGCTCAGGT AGTGGTTGTC GGGCAGCAGC ACGGGGCCGT 6361 CGCCGATGGG GGTGTTCTGC TGGTAGTGGT CGGCCAGGTG AGTCCAGGAG ATGTTTCAGC 6421 ACTGTTGCCT TTAGTCTCGA GGCAACTTAG ACAACTGAGT ATTGATCTGA GCACAGCAGG 6481 GTGTGAGCTG TTTGAAGATA CTGGGGTTGG GGGTGAAGAA ACTGCAGAGG ACTAACTGGG 6541 CTGAGACCCA GTGGCAATGT TTTAGGGCCT AAGGAATGCC TCTGAAAATC TAGATGGACA 6601 ACTTTGACTT TGAGAAAAGA GAGGTGGAAA TGAGGAAAAT GACTTTTCTT TATTAGATTT 6661 CGGTAGAAAG AACTTTCATC TTTCCCCTAT TTTTGTTATT CGTTTTAAAA CATCTATCTG 6721 GAGGCAGGAC AAGTATGGTC ATTAAAAAGA TGCAGGCAGA AGGCATATAT TGGCTCAGTC 6781 AAAGTGGGGA ACTTTGGTGG CCAAACATAC ATTGCTAAGG CTATTCCTAT ATCAGCTGGA 6841 CACATATAAA ATGCTGCTAA TGCTTCATTA CAAACTTATA TCCTTTAATT CCAGATGGGG 6901 GCAAAGTATG TCCAGGGGTG AGGAACAATT GAAACATTTG GGCTGGAGTA GATTTTGAAA 6961 GTCAGCTCTG TGTGTGTGTG TGTGTGTGTG TGTGTGAGAG CGTGTGTTTC TTTTAACGTT 7021 TTCAGCCTAC AGCATACAGG GTTCATGGTG GCAAGAAGAT AACAAGATTT AAATTATGGC 7081 CAGTGACTAG TGCTGCAAGA AGAACAACTA CCTGCATTTA ATGGGAAAGC AAAATCTCAG 7141 GCTTTGAGGG AAGTTAACAT AGGCTTGATT CTGGGTGGAA GCTGGGTGTG TAGTTATCTG 7201 GAGGCCAGGC TGGAGCTCTC AGCTCACTAT GGGTTCATCT TTATTGTCTC CTTTCATCTC 7261 AACAGCTGCA CGCTGCCGTC CTCGATGTTG TGGCGGATCT TGAAGTTCAC CTTGATGCCG 7321 TTCTTCTGCT TGTCGGCCAT GATATAGACG TTGTGGCTGT TGTAGTTGTA CTCCAGCTTG 7381 TGCCCCAGGA TGTTGCCGTC CTCCTTGAAG TCGATGCCCT TCAGCTCGAT GCGGTTCACC 7441 AGGGTGTCGC CCTCGAACTT CACCTCGGCG CGGGTCTTGT AGTTGCCGTC GTCCTTGAAG 7501 AAGATGGTGC GCTCCTGGAC GTAGCCTTCG GGCATGGCGG ACTTGAAGAA GTCGTGCTGC 7561 TTCATGTGGT CGGGGTAGCG GCTGAAGCAC TGCACGCCGT AGGTCAGGGT GGTCACGAGG 7621 GTGGGCCAGG GCACGGGCAG CTTGCCGGTG GTGCAGATGA ACTTCAGGGT CAGCTTGCCG 7681 TAGGTGGCAT CGCCCTCGCC CTCGCCGGAC ACGCTGAACT TGTGGCCGTT TACGTCGCCG 7741 TCCAGCTCGA CCAGGATGGG CACCACCCCG GTGAACAGCT CCTCGCCCTT GCTCACCATA 7801 GGGCCGGGAT TCTCCTCCAC GTCACCGCAT GTTAGAAGAC TTCCTCTGCC CTCTCTTGGA 7861 GGCAGGGCCT GCATGTGCAG GGCATCGTAG GTATCCTTGG TGGCTGTGCT CAGTCCCTGG 7921 TACAGTCCAT CGTGGCCCTT GCCTCTTCTT CTCTCGCCCT TCATGCCGAT CTCGCTGTAG 7981 GCCTCGGCCA TCTTGTCTTT CTGCAGCTCA TTATACAGGC CCTCTTGAGG ATTCTTTCTC 8041 CGCTGGGGCT TGCCGCCCAT CTCAGGATCT CTGCCTCTCC GCTTATCCAG CACGTCGTAC 8101 TCTTCTCTTC TCCCCAGGTT CAGCTCGTTG TACAGCTGAT TCTGGCCCTG CTGGTAAGCA 8161 GGAGCGTCGG CGGATCTGCT GAACTTCACT CTGCAGTACA GGGTGATGAC CAGAGAGAGC 8221 AGCAGAACGC CACATGTGCC AGCCAGAGGG GCCCAAATGT AGATATCCAG GCCTCTGGTA 8281 TGCACAGCTC CGCCAGCTGC AGGTCTACAG GCTTCAGGTC TGAGAGACAG AGGCTGGCTG 8341 GCGATTGTAG GAGCTGGTGT AGGTGGTCTA GGAGCGGGTG TTGTTGTAGG CTTGGCGGGC 8401 AGAAACACGG GCACGAAGTG GCTGAAGTAC ATGATGCTAT TGCTCAGGGC TCCGCTTCCT 8461 CCGCCTCCGC TAGAAGAAAC TGTGACCAGG GTGCCCTGTC CCCAAACATC CATGGCGTAG 8521 AAGCCGTCGC CTCCCCATCT AGAACAGTAG TACACGGCGG TGTCCTCGGC TCTCAGGCTG 8581 TTCATCTGCA GGTAGGCGGT GTTCTTGCTG GTGTCGGCGC TGATGGTGAA TCTGCCCTTC 8641 ACGCTATCGG CGTATCTGGT GTAGCCGTTG GTGGGGTAGA TTCTGGCGAC CCATTCAAGT 8701 CCCTTTCCAG GGGCCTGTCG GACCCAGTGG ATGTAGGTGT CCTTGATGTT GAAGCCGCTG 8761 GCGGCACAAG ACAGTCTCAG AGAGCCGCCA GGCTGAACAA GTCCTCCGCC AGATTCAACC 8821 AGCTGCACCT CAGATCCTTC GCCAGATCCA GGCTTTCCAG AGCCGCTGGT GCTGCCTGTT 8881 CTCTTGATTT CCACCTTGGT GCCCTGGCCA AAGGTTGGAG GTGTGGTGTA GTGCTGCTGG 8941 CAGTAGTAGG TGGCGAAGTC CTCAGGCTGC AGGCTAGAGA TGGTCAGGGT GAAGTCGGTG 9001 CCAGATCTGC TGCCGCTGAA TCTGCTTGGC ACGCCGCTGT ACAGAAAGCT GGCGCTGTAG 9061 ATCAGCAGCT TAGGGGCTTT TCCAGGCTTC TGCTGATACC AGGCCACGGC GGTATTCACA 9121 TCCTGGCTGG CTCTACAGGT GATGGTCACT CTATCGCCCA CAGAGGCAGA CAGGCTGCTA 9181 GGGCTCTGTG TCATCTGGAT GTCGCTGATG CTGCAGGCCA CTGTTCCCAG CAGCAGCAGA 9241 GACTGCAGCC ACATTCGAAG CTTGAGCTCG AGATCTGAGT CCGGTAGCGC TAGCGGATCT 9301 GACGGTTCAC TAAACCAGCT CTGCTTATAT AGACCTCCCA CCGTACACGC CTACCGCCCA 9361 TTTGCGTCAA TGGGGCGGAG TTGTTACGAC ATTTTGGAAA GTCCCGTTGA TTTTGGTGCC 9421 AAAACAAACT CCCATTGACG TCAATGGGGT GGAGACTTGG AAATCCCCGT GAGTCAAACC 9481 GCTATCCACG CCCATTGATG TACTGCCAAA ACCGCATCAC CATGGTAATA GCGATGACTA 9541 ATACGTAGAT GTACTGCCAA GTAGGAAAGT CCCATAAGGT CATGTACTGG GCATAATGCC 9601 AGGCGGGCCA TTTACCGTCA TTGACGTCAA TAGGGGGCGT ACTTGGCATA TGATACACTT 9661 GATGTACTGC CAAGTGGGCA GTTTACCGTA AATACTCCAC CCATTGACGT CAATGGAAAG 9721 TCCCTATTGG CGTTACTATG GGAACATACG TCATTATTGA CGTCAATGGG CGGGGGTCGT 9781 TGGGCGGTCA GCCAGGCGGG CCATTTACCG TAAGTTATGT AACGCGGAAC TCCATATATG 9841 GGCTATGAAC TAATGACCCC GTAATTGATT ACTATTAGCC CGGGGGATCC AGACATGATA 9901 AGATACATTG ATGAGTTTGG ACAAACCACA ACTAGAATGC AGTGAAAAAA ATGCTTTATT 9961 TGTGAAATTT GTGATGCTAT TGCTTTATTT GTAACCATTA TAAGCTGCAA TAAACAAGTT 10021 AACAACAACA ATTGCATTCA TTTTATGTTT CAGGTTCAGG GGGAGGTGTG GGAGGTTTTT 10081 TAAAGCAAGT AAAACCTCTA CAAATGTGGT ATGGCTGATT ATGATCCGGC TGCCTCGCGC 10141 GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT CCCGGAGACG GTCACAGCTT 10201 GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG CGCGTCAGCG GGTGTTGGCG 10261 GGTGTCGGGG CGCAGCCATG AGGTCGATCG ACTCTAGAGG ATCGATCCCC GCCCCGGACG 10321 AACTAAACCT GACTACGACA TCTCTGCCCC TTCTTCGCGG GGCAGTGCAT GTAATCCCTT 10381 CAGTTGGTTG GTACAACTTG CCAACTGGGC CCTGTTCCAC ATGTGACACG GGGGGGGACC 10441 AAACACAAAG GGGTTCTCTG ACTGTAGTTG ACATCCTTAT AAATGGATGT GCACATTTGC 10501 CAACACTGAG TGGCTTTCAT CCTGGAGCAG ACTTTGCAGT CTGTGGACTG CAACACAACA 10561 TTGCCTTTAT GTGTAACTCT TGGCTGAAGC TCTTACACCA ATGCTGGGGG ACATGTACCT 10621 CCCAGGGGCC CAGGAAGACT ACGGGAGGCT ACACCAACGT CAATCAGAGG GGCCTGTGTA 10681 GCTACCGATA AGCGGACCCT CAAGAGGGCA TTAGCAATAG TGTTTATAAG GCCCCCTTGT 10741 TAACCCTAAA CGGGTAGCAT ATGCTTCCCG GGTAGTAGTA TATACTATCC AGACTAACCC 10801 TAATTCAATA GCATATGTTA CCCAACGGGA AGCATATGCT ATCGAATTAG GGTTAGTAAA 10861 AGGGTCCTAA GGAACAGCGA TATCTCCCAC CCCATGAGCT GTCACGGTTT TATTTACATG 10921 GGGTCAGGAT TCCACGAGGG TAGTGAACCA TTTTAGTCAC AAGGGCAGTG GCTGAAGATC 10981 AAGGAGCGGG CAGTGAACTC TCCTGAATCT TCGCCTGCTT CTTCATTCTC CTTCGTTTAG 11041 CTAATAGAAT AACTGCTGAG TTGTGAACAG TAAGGTGTAT GTGAGGTGCT CGAAAACAAG 11101 GTTTCAGGTG ACGCCCCCAG AATAAAATTT GGACGGGGGG TTCAGTGGTG GCATTGTGCT 11161 ATGACACCAA TATAACCCTC ACAAACCCCT TGGGCAATAA ATACTAGTGT AGGAATGAAA 11221 CATTCTGAAT ATCTTTAACA ATAGAAATCC ATGGGGTGGG GACAAGCCGT AAAGACTGGA 11281 TGTCCATCTC ACACGAATTT ATGGCTATGG GCAACACATA ATCCTAGTGC AATATGATAC 11341 TGGGGTTATT AAGATGTGTC CCAGGCAGGG ACCAAGACAG GTGAACCATG TTGTTACACT 11401 CTATTTGTAA CAAGGGGAAA GAGAGTGGAC GCCGACAGCA GCGGACTCCA CTGGTTGTCT 11461 CTAACACCCC CGAAAATTAA ACGGGGCTCC ACGCCAATGG GGCCCATAAA CAAAGACAAG 11521 TGGCCACTCT TTTTTTTGAA ATTGTGGAGT GGGGGCACGC GTCAGCCCCC ACACGCCGCC 11581 CTGCGGTTTT GGACTGTAAA ATAAGGGTGT AATAACTTGG CTGATTGTAA CCCCGCTAAC 11641 CACTGCGGTC AAACCACTTG CCCACAAAAC CACTAATGGC ACCCCGGGGA ATACCTGCAT 11701 AAGTAGGTGG GCGGGCCAAG ATAGGGGCGC GATTGCTGCG ATCTGGAGGA CAAATTACAC 11761 ACACTTGCGC CTGAGCGCCA AGCACAGGGT TGTTGGTCCT CATATTCACG AGGTCGCTGA 11821 GAGCACGGTG GGCTAATGTT GCCATGGGTA GCATATACTA CCCAAATATC TGGATAGCAT 11881 ATGCTATCCT AATCTATATC TGGGTAGCAT AGGCTATCCT AATCTATATC TGGGTAGCAT 11941 ATGCTATCCT AATCTATATC TGGGTAGTAT ATGCTATCCT AATTTATATC TGGGTAGCAT 12001 AGGCTATCCT AATCTATATC TGGGTAGCAT ATGCTATCCT AATCTATATC TGGGTAGTAT 12061 ATGCTATCCT AATCTGTATC CGGGTAGCAT ATGCTATCCT AATAGAGATT AGGGTAGTAT 12121 ATGCTATCCT AATTTATATC TGGGTAGCAT ATACTACCCA AATATCTGGA TAGCATATGC 12181 TATCCTAATC TATATCTGGG TAGCATATGC TATCCTAATC TATATCTGGG TAGCATAGGC 12241 TATCCTAATC TATATCTGGG TAGCATATGC TATCCTAATC TATATCTGGG TAGTATATGC 12301 TATCCTAATT TATATCTGGG TAGCATAGGC TATCCTAATC TATATCTGGG TAGCATATGC 12361 TATCCTAATC TATATCTGGG TAGTATATGC TATCCTAATC TGTATCCGGG TAGCATATGC 12421 TATCCTCATG CATATACAGT CAGCATATGA TACCCAGTAG TAGAGTGGGA GTGCTATCCT 12481 TTGCATATGC CGCCACCTCC CAAGGGGGCG TGAATTTTCG CTGCTTGTCC TTTTCCTGCA 12541 TGCTGGTTGC TCCCATTCTT AGGTGAATTT AAGGAGGCCA GGCTAAAGCC GTCGCATGTC 12601 TGATTGCTCA CCAGGTAAAT GTCGCTAATG TTTTCCAACG CGAGAAGGTG TTGAGCGCGG 12661 AGCTGAGTGA CGTGACAACA TGGGTATGCC CAATTGCCCC ATGTTGGGAG GACGAAAATG 12721 GTGACAAGAC AGATGGCCAG AAATACACCA ACAGCACGCA TGATGTCTAC TGGGGATTTA 12781 TTCTTTAGTG CGGGGGAATA CACGGCTTTT AATACGATTG AGGGCGTCTC CTAACAAGTT 12841 ACATCACTCC TGCCCTTCCT CACCCTCATC TCCATCACCT CCTTCATCTC CGTCATCTCC 12901 GTCATCACCC TCCGCGGCAG CCCCTTCCAC CATAGGTGGA AACCAGGGAG GCAAATCTAC 12961 TCCATCGTCA AAGCTGCACA CAGTCACCCT GATATTGCAG GTAGGAGCGG GCTTTGTCAT 13021 AACAAGGTCC TTAATCGCAT CCTTCAAAAC CTCAGCAAAT ATATGAGTTT GTAAAAAGAC 13081 CATGAAATAA CAGACAATGG ACTCCCTTAG CGGGCCAGGT TGTGGGCCGG GTCCAGGGGC 13141 CATTCCAAAG GGGAGACGAC TCAATGGTGT AAGACGACAT TGTGGAATAG CAAGGGCAGT 13201 TCCTCGCCTT AGGTTGTAAA GGGAGGTCTT ACTACCTCCA TATACGAACA CACCGGCGAC 13261 CCAAGTTCCT TCGTCGGTAG TCCTTTCTAC GTGACTCCTA GCCAGGAGAG CTCTTAAACC 13321 TTCTGCAATG TTCTCAAATT TCGGGTTGGA ACCTCCTTGA CCACGATGCT TTCCAAACCA 13381 CCCTCCTTTT TTGCGCCTGC CTCCATCACC CTGACCCCGG GGTCCAGTGC TTGGGCCTTC 13441 TCCTGGGTCA TCTGCGGGGC CCTGCTCTAT CGCTCCCGGG GGCACGTCAG GCTCACCATC 13501 TGGGCCACCT TCTTGGTGGT ATTCAAAATA ATCGGCTTCC CCTACAGGGT GGAAAAATGG 13561 CCTTCTACCT GGAGGGGGCC TGCGCGGTGG AGACCCGGAT GATGATGACT GACTACTGGG 13621 ACTCCTGGGC CTCTTTTCTC CACGTCCACG ACCTCTCCCC CTGGCTCTTT CACGACTTCC 13681 CCCCCTGGCT CTTTCACGTC CTCTACCCCG GCGGCCTCCA CTACCTCCTC GACCCCGGCC 13741 TCCACTACCT CCTCGACCCC GGCCTCCACT GCCTCCTCGA CCCCGGCCTC CACCTCCTGC 13801 TCCTGCCCCT CCTGCTCCTG CCCCTCCTCC TGCTCCTGCC CCTCCTGCCC CTCCTGCTCC 13861 TGCCCCTCCT GCCCCTCCTG CTCCTGCCCC TCCTGCCCCT CCTGCTCCTG CCCCTCCTGC 13921 CCCTCCTCCT GCTCCTGCCC CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC CTGCCCCTCC 13981 TGCTCCTGCC CCTCCTGCCC CTCCTGCTCC TGCCCCTCCT GCCCCTCCTG CTCCTGCCCC 14041 TCCTGCTCCT GCCCCTCCTG CTCCTGCCCC TCCTGCTCCT GCCCCTCCTG CCCCTCCTGC 14101 CCCTCCTCCT GCTCCTGCCC CTCCTGCTCC TGCCCCTCCT GCCCCTCCTG CCCCTCCTGC 14161 TCCTGCCCCT CCTCCTGCTC CTGCCCCTCC TGCCCCTCCT GCCCCTCCTC CTGCTCCTGC 14221 CCCTCCTGCC CCTCCTCCTG CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC CTGCCCCTCC 14281 TGCCCCTCCT CCTGCTCCTG CCCCTCCTGC CCCTCCTCCT GCTCCTGCCC CTCCTCCTGC 14341 TCCTGCCCCT CCTGCCCCTC CTGCCCCTCC TCCTGCTCCT GCCCCTCCTC CTGCTCCTGC 14401 CCCTCCTGCC CCTCCTGCCC CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC CTCCTGCTCC 14461 TGCCCCTCCT GCTCCTGCCC CTCCCGCTCC TGCTCCTGCT CCTGTTCCAC CGTGGGTCCC 14521 TTTGCAGCCA ATGCAACTTG GACGTTTTTG GGGTCTCCGG ACACCATCTC TATGTCTTGG 14581 CCCTGATCCT GAGCCGCCCG GGGCTCCTGG TCTTCCGCCT CCTCGTCCTC GTCCTCTTCC 14641 CCGTCCTCGT CCATGGTTAT CACCCCCTCT TCTTTGAGGT CCACTGCCGC CGGAGCCTTC 14701 TGGTCCAGAT GTGTCTCCCT TCTCTCCTAG GCCATTTCCA GGTCCTGTAC CTGGCCCCTC 14761 GTCAGACATG ATTCACACTA AAAGAGATCA ATAGACATCT TTATTAGACG ACGCTCAGTG 14821 AATACAGGGA GTGCAGACTC CTGCCCCCTC CAACAGCCCC CCCACCCTCA TCCCCTTCAT 14881 GGTCGCTGTC AGACAGATCC AGGTCTGAAA ATTCCCCATC CTCCGAACCA TCCTCGTCCT 14941 CATCACCAAT TACTCGCAGC CCGGAAAACT CCCGCTGAAC ATCCTCAAGA TTTGCGTCCT 15001 GAGCCTCAAG CCAGGCCTCA AATTCCTCGT CCCCCTTTTT GCTGGACGGT AGGGATGGGG 15061 ATTCTCGGGA CCCCTCCTCT TCCTCTTCAA GGTCACCAGA CAGAGATGCT ACTGGGGCAA 15121 CGGAAGAAAA GCTGGGTGCG GCCTGTGAGG ATCAGCTTAT CGATGATAAG CTGTCAAACA 15181 TGAGAATTCT TGAAGACGAA AGGGCCTCGT GATACGCCTA TTTTTATAGG TTAATGTCAT 15241 GATAATAATG GTTTCTTAGA CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC 15301 TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG 15361 ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC 15421 CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT 15481 GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG GGTTACATCG AACTGGATCT 15541 CAACAGCGGT AAGATCCTTG AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC 15601 TTTTAAAGTT CTGCTATGTG GCGCGGTATT ATCCCGTGTT GACGCCGGGC AAGAGCAACT 15661 CGGTCGCCGC ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA 15721 GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA 15781 TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT 15841 TTTGCACAAC ATGGGGGATC ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA 15901 AGCCATACCA AACGACGAGC GTGACACCAC GATGCCTGCA GCAATGGCAA CAACGTTGCG 15961 CAAACTATTA ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT 16021 GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT 16081 TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC 16141 AGATGGTAAG CCCTCCCGTA TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA 16201 TGAACGAAAT AGACAGATCG CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC 16261 AGACCAAGTT TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG 16321 GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC 16381 GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT 16441 TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT 16501 GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT 16561 ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC 16621 ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA 16681 GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG 16741 CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG 16801 ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG 16861 GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA 16921 CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT 16981 GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG 17041 GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC 17101 TGTGGATAAC CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC 17161 CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC GGAAGAGCGC CTGATGCGGT ATTTTCTCCT 17221 TACGCATCTG TGCGGTATTT CACACCGCAT ATGGTGCACT CTCAGTACAA TCTGCTCTGA 17281 TGCCGCATAG TTAAGCCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG 17341 CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG 17401 AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC 17461 AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA 17521 TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCGGC 17581 CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA 17641 GCTTGCATGC CTGCAGGTCG GCCGCCACGA CCGGTGCCGC CACCATCCCC TGACCCACGC 17701 CCCTGACCCC TCACAAGGAG ACGACCTTCC ATGACCGAGT ACAAGCCCAC GGTGCGCCTC 17761 GCCACCCGCG ACGACGTCCC CCGGGCCGTA CGCACCCTCG CCGCCGCGTT CGCCGACTAC 17821 CCCGCCACGC GCCACACCGT CGACCCGGAC CGCCACATCG AGCGGGTCAC CGAGCTGCAA 17881 GAACTCTTCC TCACGCGCGT CGGGCTCGAC ATCGGCAAGG TGTGGGTCGC GGACGACGGC 17941 GCCGCGGTGG CGGTCTGGAC CACGCCGGAG AGCGTCGAAG CGGGGGCGGT GTTCGCCGAG 18001 ATCGGCCCGC GCATGGCCGA GTTGAGCGGT TCCCGGCTGG CCGCGCAGCA ACAGATGGAA 18061 GGCCTCCTGG CGCCGCACCG GCCCAAGGAG CCCGCGTGGT TCCTGGCCAC CGTCGGCGTC 18121 TCGCCCGACC ACCAGGGCAA GGGTCTGGGC AGCGCCGTCG TGCTCCCCGG AGTGGAGGCG 18181 GCCGAGCGCG CCGGGGTGCC CGCCTTCCTG GAGACCTCCG CGCCCCGCAA CCTCCCCTTC 18241 TACGAGCGGC TCGGCTTCAC CGTCACCGCC GACGTCGAGG TGCCCGAAGG ACCGCGCACC 18301 TGGTGCATGA CCCGCAAGCC CGGTGCCTGA CGCCCGCCCC ACGACCCGCA GCGCCCGACC 18361 GAAAGGAGCG CACGACCCCA TGGCTCCGAC CGAAGCCGAC CCGGGCGGCC CCGCCGACCC 18421 CGCACCCGCC CCCGAGGCCC ACCGACTCTA GAGGATCATA ATCAGCCATA CCACATTTGT 18481 AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT 18541 GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA 18601 TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC 18661 CAAACTCATC AATGTATCTT ATCATGTCTG GATCACTCGC CGATAGTGGA AACCGACGCC 18721 CCAGCACTCG TCCGAGGGCA AAGGAATAGG GGAGATGGGG GAGGCTAACT GAAACACGGA 18781 AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT AAAAAGACAG AATAAAACGC 18841 ACGGGTGTTG GGTCGTTTGT TCATAAACGC GGGGTTCGGT CCCAGGGCTG GCACTCTGTC 18901 GATACCCCAC CGAGACCCCA TTGGGGCCAA TACGCCCGCG TTTCTTCCTT TTCCCCACCC 18961 CACCCCCCAA GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT 19021 GCCATAGCCA CTGGCCCCGT GGGTTAGGGA CGGGGTCCCC CATGGGGAAT GGTTTATGGT 19081 TCGTGGGGGT TATTATTTTG GGCGTTGCGT GGGGTCTGGT CCACGACTGG ACTGAGCAGA 19141 CAGACCCATG GTTTTTGGAT GGCCTGGGCA TGGACCGCAT GTACTGGCGC GACACGAACA 19201 CCGGGCGTCT GTGGCTGCCA AACACCCCCG ACCCCCAAAA ACCACCGCGC GGATTTCTGG 19261 CGTGCCAAGC TAGTCGACCA ATTCTCATGT TTGACAGCTT ATCATCGCAG ATCCGGGCAA 19321 CGTTGTTGCA TTGCTGCAGG CGCAGAACTG GTAGGTATGG AAGATCTCTA GAAGCTGGGT 19381 ACCAGCTGCT AGCAAGCTTG CTAGCGGCCG GCTCGAGTTT ACTCCCTATC AGTGATAGAG 19441 AACGTATGTC GAGTTTACTC CCTATCAGTG ATAGAGAACG ATGTCGAGTT TACTCCCTAT 19501 CAGTGATAGA GAACGTATGT CGAGTTTACT CCCTATCAGT GATAGAGAAC GTATGTCGAG 19561 TTTACTCCCT ATCAGTGATA GAGAACGTAT GTCGAGTTTA TCCCTATCAG TGATAGAGAA 19621 CGTATGTCGA GTTTACTCCC TATCAGTGAT AGAGAACGTA TGTCGAGGTA GGCGTGTACG 19681 GTGGGAGGCC TATATAAGCA GAGCTCGTTT AGTGAACCGT CAGATCGCCG (SEQ ID NO: 41) LINE-1 ORF2-NLS mRNA (SEQ ID NO: 42) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGCCG TCAGACCATC AAGACTAGGA 1081 AGAAACTGCA TCAACTAATG AGCAAAATCA CCAGCTAACA TCATAGTATA CATGACCGGC 1141 TCTAACTCAC ATATCACCAT CCTTACACTT AACATTAACG GCCTCAACTC AGCTATCAAG 1201 CGCCATCGGC TGGCCAGCTG GATCAAATCA CAGGATCCAA GCGTTTGTTG CATCCAAGAG 1261 ACCCACCTGA CCTGTAGAGA TACTCACCGC CTCAAGATCA AGGGATGGCG AAAGATTTAT 1321 CAGGCGAACG GTAAGCAGAA GAAAGCCGGA GTCGCAATTC TGGTCTCAGA CAAGACGGAT 1381 TTCAAGCCCA CCAAAATTAA GCGTGATAAG GAAGGTCACT ATATTATGGT GAAAGGCAGC 1441 ATACAGCAGG AAGAACTTAC CATATTGAAC ATCTACGCGC CAAACACCGG CGCACCTCGC 1501 TTTATCAAAC AGGTCCTGTC CGATCTGCAG CGAGATCTGG ATTCTCATAC GTTGATTATG 1561 GGTGATTTCA ATACACCATT GAGCACCCTG GATCGCAGCA CCAGGCAAAA GGTAAATAAA 1621 GACACGCAAG AGCTCAATAG CGCACTGCAT CAGGCAGATC TCATTGATAT TTATCGCACT 1681 CTTCATCCTA AGAGTACCGA GTACACATTC TTCAGCGCCC CACATCATAC ATACTCAAAG 1741 ATCGATCATA TCGTCGGCTC AAAGGCTCTG CTGTCAAAGT GCAAGCGCAC AGAGATAATT 1801 ACAAATTACC TGTCAGATCA TAGCGCGATC AAGCTCGAGC TGAGAATCAA GAACCTGACC 1861 CAGAGCCGGA GTACCACTTG GAAGCTTAAT AACCTGCTGC TCAACGATTA TTGGGTCCAC 1921 AATGAGATGA AGGCAGAGAT TAAAATGTTC TTCGAAACAA ATGAGAATAA GGATACTACC 1981 TATCAAAACC TTTGGGATGC CTTTAAGGCC GTCTGCAGAG GCAAGTTCAT CGCCCTCAAC 2041 GCCTATAAAA GAAAACAAGA GAGATCTAAG ATCGATACTC TCACCTCTCA GCTGAAGGAG 2101 TTGGAGAAAC AGGAACAGAC CCACTCCAAG GCGTCAAGAC GGCAGGAGAT CACAAAGATT 2161 CGCGCCGAGT TGAAAGAGAT CGAAACCCAA AAGACTCTTC AGAAAATTAA CGAGTCTCGT 2221 AGTTGGTTCT TCGAGCGGAT TAATAAGATA GACAGACCTC TGGCACGACT GATTAAGAAG 2281 AAGCGCGAAA AGAACCAGAT TGATACCATC AAGAACGACA AGGGCGACAT CACTACTGAC 2341 CCGACCGAGA TCCAGACCAC TATTCGGGAG TATTATAAGC ATTTGTATGC TAACAAGCTT 2401 GAGAACCTGG AAGAGATGGA CACTTTTCTG GATACCTATA CTCTGCCACG GCTTAATCAA 2461 GAGGAAGTCG AGTCCCTCAA CCGCCCAATT ACAGGAAGCG AGATTGTGGC CATAATTAAC 2521 TCCCTGCCGA CAAAGAAATC TCCTGGTCCG GACGGGTTTA CAGCTGAGTT TTATCAACGG 2581 TATATGGAAG AGCTTGTACC GTTTCTGCTC AAGCTCTTTC AGTCTATAGA AAAGGAAGGC 2641 ATCTTGCCCA ATTCCTTCTA CGAAGCTTCT ATAATACTTA TTCCCAAACC AGGACGCGAT 2701 ACCACAAAGA AGGAAAACTT CCGGCCCATT AGTCTCATGA ATATCGACGC TAAAATATTG 2761 AACAAGATTC TCGCCAACAG AATCCAACAA CATATTAAGA AATTGATACA TCACGACCAG 2821 GTGGGGTTTA TACCTGGCAT GCAGGGCTGG TTTAACATCC GGAAGAGTAT TAACGTCATT 2881 CAACACATTA ATAGAGCTAA GGATAAGAAT CATATGATCA TCTCTATAGA CGCGGAAAAG 2941 GCATTCGATA AGATTCAGCA GCCATTTATG CTCAAGACTC TGAACAAACT CGGCATCGAC 3001 GGAACATATT TTAAGATTAT TCGCGCAATT TACGATAAGC CGACTGCTAA CATTATCCTT 3061 AACGGCCAAA AGCTCGAGGC CTTTCCGCTC AAGACTGGAA CCCGCCAAGG CTGTCCCCTC 3121 TCCCCGCTTT TGTTTAATAT TGTACTCGAG GTGCTGGCTA GGGCTATTCG TCAAGAGAAA 3181 GAGATTAAAG GGATACAGCT CGGGAAGGAA GAGGTCAAGC TTTCCTTGTT CGCCGATGAT 3241 ATGATTGTGT ACCTGGAGAA TCCTATTGTG TCTGCTCAGA ACCTTCTTAA ACTTATTTCT 3301 AACTTTAGCA AGGTCAGCGG CTATAAGATT AACGTCCAGA AATCTCAGGC CTTTCTGTAC 3361 ACAAATAATC GACAGACCGA ATCCCAGATA ATGGGTGAGC TTCCGTTTGT CATAGCCAGC 3421 AAAAGGATAA AGTATCTCGG AATCCAGCTG ACACGAGACG TTAAAGATTT GTTTAAGGAA 3481 AATTACAAGC CTCTCCTGAA AGAGATTAAG GAAGATACTA ATAAGTGGAA GAATATCCCC 3541 TGTTCATGGG TTGGCAGAAT CAACATAGTG AAGATGGCAA TACTTCCTAA AGTGATATAT 3601 CGCTTTAACG CCATCCCAAT TAAACTGCCT ATGACCTTCT TTACGGAGCT CGAGAAAACA 3661 ACCCTTAAAT TTATATGGAA TCAAAAGAGA GCAAGAATAG CGAAGTCCAT CTTGAGCCAG 3721 AAGAATAAGG CCGGTGGGAT TACTTTGCCT GATTTTAAGT TGTATTATAA AGCCACAGTA 3781 ACTAAGACAG CCTGGTATTG GTATCAGAAT AGAGACATCG ACCAGTGGAA TCGGACCGAA 3841 CCATCAGAGA TAATGCCCCA CATCTATAAT TACCTTATAT TCGATAAGCC AGAAAAGAAT 3901 AAACAGTGGG GCAAAGACAG CCTCTTCAAC AAGTGGTGTT GGGAGAATTG GCTGGCCATA 3961 TGCCGGAAAC TCAAGCTCGA CCCCTTTCTT ACACCCTACA CTAAAATCAA CAGTAGGTGG 4021 ATCAAGGACT TGAATGTCAA GCCAAAGACT ATAAAGACAC TGGAAGAGAA TCTTGGGATC 4081 ACAATACAAG ATATAGGCGT CGGCAAAGAT TTTATGTCAA AGACGCCCAA GGCCATGGCC 4141 ACTAAGGATA AGATTGATAA GTGGGACCTT ATTAAGCTCA AAAGCTTCTG TACTGCCAAG 4201 GAGACCACGA TCAGAGTTAA TAGGCAGCCC ACTACATGGG AAAAGATTTT CGCCACTTAT 4261 TCATCAGATA AGGGGTTGAT AAGCAGAATA TATAACGAGC TGAAGCAGAT CTACAAGAAG 4321 AAAACGAATA ATCCCATCAA GAAGTGGGCA AAAGATATGA ACAGGCATTT TAGCAAAGAG 4381 GATATCTACG CCGCGAAGAA GCATATGAAG AAGTGTAGTT CAAGCTTGGC CATTCGTGAG 4441 ATGCAGATTA AGACGACCAT GCGATACCAC CTTACCCCAG TGAGGATGGC AATTATCAAG 4501 AAATCTGGCA ATAATAGATG TTGGCGGGGC TGTGGCGAGA TTGGCACCCT GCTCCATTGC 4561 TGGTGGGATT GCAAGCTGGT GCAGCCGCTT TGGAAATCAG TCTGGCGCTT TCTGAGGGAC 4621 CTCGAGCTTG AGATTCCCTT CGATCCCGCA ATTCCCTTGC TCGGAATCTA TCCTAACGAA 4681 TACAAGAGCT GTTGTTACAA GGATACGTGT ACCCGGATGT TCATCGCGGC CTTGTTTACG 4741 ATAGCTAAGA CGTGGAATCA GCCTAAGTGC CCCACAATGA TCGATTGGAT CAAGAAAATG 4801 TGGCATATTT ATACCATGGA GTATTACGCA GCAATTAAGA ATGACGAATT TATTTCCTTC 4861 GTTGGGACCT GGATGAAGCT GGAGACTATT ATTCTGAGCA AGCTGTCTCA GGAGCAAAAG 4921 ACAAAGCATA GAATCTTCTC TCTCATTGGT GGTAACGACT ACAAAGACGA TGACGACAAG 4981 CCCGCCGCCA AGAGGGTGAA GCTGGACTAA AGCGCTTCTA GAAGTTGTCT CCTCCTGCAC 5041 TGACTGACTG ATACAATCGA TTTCTGGATC CGCAGGCCTA ATCAACCTCT GGATTACAAA 5101 ATTTGTGAAA GATTGACTGG TATTCTTAAC TATGTTGCTC CTTTTACGCT ATGTGGATAC 5161 GCTGCTTTAA TGCCTTTGTA TCATGCTATT GCTTCCCGTA TGGCTTTCAT TTTCTCCTCC 5221 TTGTATAAAT CCTGGTTGCT GTCTCTTTAT GAGGAGTTGT GGCCCGTTGT CAGGCAACGT 5281 GGCGTGGTGT GCACTGTGTT TGCTGACGCA ACCCCCACTG GTTGGGGCAT TGCCACCACC 5341 TGTCAGCTCC TTTCCGGGAC TTTCGCTTTC CCCCTCCCTA TTGCCACGGC GGAACTCATC 5401 GCCGCCTGCC TTGCCCGCTG CTGGACAGGG GCTCGGCTGT TGGGCACTGA CAATTCCGTG 5461 GTGTTGTCGG GGAAGCTGAC GTCCTTTCCA TGGCTGCTCG CCTGTGTTGC CACCTGGATT 5521 CTGCGCGGGA CGTCCTTCTG CTACGTCCCT TCGGCCCTCA ATCCAGCGGA CCTTCCTTCC 5581 CGCTGAGAGA CACAAAAAAT TCCAACACAC TATTGCAATG AAAATAAATT TCCTTTATTA 5641 GCCAGAAGTC AGATGCTCAA GGGGCTTCAT GATGTCCCCA TAATTTTTGG CAGAGGGAAA 5701 AAGATCTCAG TGGTATTTGT GAGCCAGGGC ATTGGCCTTC TGATAGGCAG CCTGCACCTG 5761 AGGAGTGCGG CCGCTTTACT TGTACAGCTC GTCCATGCCG AGAGTGATCC CGGCGGCGGT 5821 CACGAACTCC AGCAGGACCA TGTGATCGCG CTTCTCGTTG GGGTCTTTGC TCAGGGCGGA 5881 CTGGGTGCTC AGGTAGTGGT TGTCGGGCAG CAGCACGGGG CCGTCGCCGA TGGGGGTGTT 5941 CTGCTGGTAG TGGTCGGCGA GCTGCACGCT GCCGTCCTCG ATGTTGTGGC GGATCTTGAA 6001 GTTCACCTTG ATGCCGTTCT TCTGCTTGTC GGCCATGATA TAGACGTTGT GGCTGTTGTA 6061 GTTGTACTCC AGCTTGTGCC CCAGGATGTT GCCGTCCTCC TTGAAGTCGA TGCCCTTCAG 6121 CTCGATGCGG TTCACCAGGG TGTCGCCCTC GAACTTCACC TCGGCGCGGG TCTTGTAGTT 6181 GCCGTCGTCC TTGAAGAAGA TGGTGCGCTC CTGGACGTAG CCTTCGGGCA TGGCGGACTT 6241 GAAGAAGTCG TGCTGCTTCA TGTGGTCGGG GTAGCGGCTG AAGCACTGCA CGCCGTAGGT 6301 CAGGGTGGTC ACGAGGGTGG GCCAGGGCAC GGGCAGCTTG CCGGTGGTGC AGATGAACTT 6361 CAGGGTCAGC TTGCCGTAGG TGGCATCGCC CTCGCCCTCG CCGGACACGC TGAACTTGTG 6421 GCCGTTTACG TCGCCGTCCA GCTCGACCAG GATGGGCACC ACCCCGGTGA ACAGCTCCTC 6481 GCCCTTGCTC ACCATGGTGG CGGGATCTGA CGGTTCACTA AACCAGCTCT GCTTATATAG 6541 ACCTCCCACC GTACACGCCT ACCGCCCATT TGCGTCAATG GGGCGGAGTT GTTACGACAT 6601 TTTGGAAAGT CCCGTTGATT TTGGTGCCAA AACAAACTCC CATTGACGTC AATGGGGTGG 6661 AGACTTGGAA ATCCCCGTGA GTCAAACCGC TATCCACGCC CATTGATGTA CTGCCAAAAC 6721 CGCATCACCA TGGTAATAGC GATGACTAAT ACGTAGATGT ACTGCCAAGT AGGAAAGTCC 6781 CATAAGGTCA TGTACTGGGC ATAATGCCAG GCGGGCCATT TACCGTCATT GACGTCAATA 6841 GGGGGCGTAC TTGGCATATG ATACACTTGA TGTACTGCCA AGTGGGCAGT TTACCGTAAA 6901 TACTCCACCC ATTGACGTCA ATGGAAAGTC CCTATTGGCG TTACTATGGG AACATACGTC 6961 ATTATTGACG TCAATGGGCG GGGGTCGTTG GGCGGTCAGC CAGGCGGGCC ATTTACCGTA 7021 AGTTATGTAA CGGGCCTGCT GCCGGCTCTG CGGCCTCTTC CGCGTCTTCG CCTTCGCCCT 7081 CAGACGAGTC GGATCTCCCT TTGGGCCGCC TCCCCGCCTG TCTAGCTTGA CTGACTGAGA 7141 TACAGCGTAC CTTCAGCTCA CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC 7201 AACTAGAATG CAGTGAAAAA AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT 7261 TGTAACCATT ATAAGCTGCA ATAAACAAGT T (SEQ ID NO: 42) LINE-1 alu mRNA GFP (SEQ ID NO: 43) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGCCG TCAGACCATC AAGACTAGGA 1081 AGAAACTGCA TCAACTAATG AGCAAAATCA CCAGCTAACA TCATAGTATA CATGACCGGC 1141 TCTAACTCAC ATATCACCAT CCTTACACTT AACATTAACG GCCTCAACTC AGCTATCAAG 1201 CGCCATCGGC TGGCCAGCTG GATCAAATCA CAGGATCCAA GCGTTTGTTG CATCCAAGAG 1261 ACCCACCTGA CCTGTAGAGA TACTCACCGC CTCAAGATCA AGGGATGGCG AAAGATTTAT 1321 CAGGCGAACG GTAAGCAGAA GAAAGCCGGA GTCGCAATTC TGGTCTCAGA CAAGACGGAT 1381 TTCAAGCCCA CCAAAATTAA GCGTGATAAG GAAGGTCACT ATATTATGGT GAAAGGCAGC 1441 ATACAGCAGG AAGAACTTAC CATATTGAAC ATCTACGCGC CAAACACCGG CGCACCTCGC 1501 TTTATCAAAC AGGTCCTGTC CGATCTGCAG CGAGATCTGG ATTCTCATAC GTTGATTATG 1561 GGTGATTTCA ATACACCATT GAGCACCCTG GATCGCAGCA CCAGGCAAAA GGTAAATAAA 1621 GACACGCAAG AGCTCAATAG CGCACTGCAT CAGGCAGATC TCATTGATAT TTATCGCACT 1681 CTTCATCCTA AGAGTACCGA GTACACATTC TTCAGCGCCC CACATCATAC ATACTCAAAG 1741 ATCGATCATA TCGTCGGCTC AAAGGCTCTG CTGTCAAAGT GCAAGCGCAC AGAGATAATT 1801 ACAAATTACC TGTCAGATCA TAGCGCGATC AAGCTCGAGC TGAGAATCAA GAACCTGACC 1861 CAGAGCCGGA GTACCACTTG GAAGCTTAAT AACCTGCTGC TCAACGATTA TTGGGTCCAC 1921 AATGAGATGA AGGCAGAGAT TAAAATGTTC TTCGAAACAA ATGAGAATAA GGATACTACC 1981 TATCAAAACC TTTGGGATGC CTTTAAGGCC GTCTGCAGAG GCAAGTTCAT CGCCCTCAAC 2041 GCCTATAAAA GAAAACAAGA GAGATCTAAG ATCGATACTC TCACCTCTCA GCTGAAGGAG 2101 TTGGAGAAAC AGGAACAGAC CCACTCCAAG GCGTCAAGAC GGCAGGAGAT CACAAAGATT 2161 CGCGCCGAGT TGAAAGAGAT CGAAACCCAA AAGACTCTTC AGAAAATTAA CGAGTCTCGT 2221 AGTTGGTTCT TCGAGCGGAT TAATAAGATA GACAGACCTC TGGCACGACT GATTAAGAAG 2281 AAGCGCGAAA AGAACCAGAT TGATACCATC AAGAACGACA AGGGCGACAT CACTACTGAC 2341 CCGACCGAGA TCCAGACCAC TATTCGGGAG TATTATAAGC ATTTGTATGC TAACAAGCTT 2401 GAGAACCTGG AAGAGATGGA CACTTTTCTG GATACCTATA CTCTGCCACG GCTTAATCAA 2461 GAGGAAGTCG AGTCCCTCAA CCGCCCAATT ACAGGAAGCG AGATTGTGGC CATAATTAAC 2521 TCCCTGCCGA CAAAGAAATC TCCTGGTCCG GACGGGTTTA CAGCTGAGTT TTATCAACGG 2581 TATATGGAAG AGCTTGTACC GTTTCTGCTC AAGCTCTTTC AGTCTATAGA AAAGGAAGGC 2641 ATCTTGCCCA ATTCCTTCTA CGAAGCTTCT ATAATACTTA TTCCCAAACC AGGACGCGAT 2701 ACCACAAAGA AGGAAAACTT CCGGCCCATT AGTCTCATGA ATATCGACGC TAAAATATTG 2761 AACAAGATTC TCGCCAACAG AATCCAACAA CATATTAAGA AATTGATACA TCACGACCAG 2821 GTGGGGTTTA TACCTGGCAT GCAGGGCTGG TTTAACATCC GGAAGAGTAT TAACGTCATT 2881 CAACACATTA ATAGAGCTAA GGATAAGAAT CATATGATCA TCTCTATAGA CGCGGAAAAG 2941 GCATTCGATA AGATTCAGCA GCCATTTATG CTCAAGACTC TGAACAAACT CGGCATCGAC 3001 GGAACATATT TTAAGATTAT TCGCGCAATT TACGATAAGC CGACTGCTAA CATTATCCTT 3061 AACGGCCAAA AGCTCGAGGC CTTTCCGCTC AAGACTGGAA CCCGCCAAGG CTGTCCCCTC 3121 TCCCCGCTTT TGTTTAATAT TGTACTCGAG GTGCTGGCTA GGGCTATTCG TCAAGAGAAA 3181 GAGATTAAAG GGATACAGCT CGGGAAGGAA GAGGTCAAGC TTTCCTTGTT CGCCGATGAT 3241 ATGATTGTGT ACCTGGAGAA TCCTATTGTG TCTGCTCAGA ACCTTCTTAA ACTTATTTCT 3301 AACTTTAGCA AGGTCAGCGG CTATAAGATT AACGTCCAGA AATCTCAGGC CTTTCTGTAC 3361 ACAAATAATC GACAGACCGA ATCCCAGATA ATGGGTGAGC TTCCGTTTGT CATAGCCAGC 3421 AAAAGGATAA AGTATCTCGG AATCCAGCTG ACACGAGACG TTAAAGATTT GTTTAAGGAA 3481 AATTACAAGC CTCTCCTGAA AGAGATTAAG GAAGATACTA ATAAGTGGAA GAATATCCCC 3541 TGTTCATGGG TTGGCAGAAT CAACATAGTG AAGATGGCAA TACTTCCTAA AGTGATATAT 3601 CGCTTTAACG CCATCCCAAT TAAACTGCCT ATGACCTTCT TTACGGAGCT CGAGAAAACA 3661 ACCCTTAAAT TTATATGGAA TCAAAAGAGA GCAAGAATAG CGAAGTCCAT CTTGAGCCAG 3721 AAGAATAAGG CCGGTGGGAT TACTTTGCCT GATTTTAAGT TGTATTATAA AGCCACAGTA 3781 ACTAAGACAG CCTGGTATTG GTATCAGAAT AGAGACATCG ACCAGTGGAA TCGGACCGAA 3841 CCATCAGAGA TAATGCCCCA CATCTATAAT TACCTTATAT TCGATAAGCC AGAAAAGAAT 3901 AAACAGTGGG GCAAAGACAG CCTCTTCAAC AAGTGGTGTT GGGAGAATTG GCTGGCCATA 3961 TGCCGGAAAC TCAAGCTCGA CCCCTTTCTT ACACCCTACA CTAAAATCAA CAGTAGGTGG 4021 ATCAAGGACT TGAATGTCAA GCCAAAGACT ATAAAGACAC TGGAAGAGAA TCTTGGGATC 4081 ACAATACAAG ATATAGGCGT CGGCAAAGAT TTTATGTCAA AGACGCCCAA GGCCATGGCC 4141 ACTAAGGATA AGATTGATAA GTGGGACCTT ATTAAGCTCA AAAGCTTCTG TACTGCCAAG 4201 GAGACCACGA TCAGAGTTAA TAGGCAGCCC ACTACATGGG AAAAGATTTT CGCCACTTAT 4261 TCATCAGATA AGGGGTTGAT AAGCAGAATA TATAACGAGC TGAAGCAGAT CTACAAGAAG 4321 AAAACGAATA ATCCCATCAA GAAGTGGGCA AAAGATATGA ACAGGCATTT TAGCAAAGAG 4381 GATATCTACG CCGCGAAGAA GCATATGAAG AAGTGTAGTT CAAGCTTGGC CATTCGTGAG 4441 ATGCAGATTA AGACGACCAT GCGATACCAC CTTACCCCAG TGAGGATGGC AATTATCAAG 4501 AAATCTGGCA ATAATAGATG TTGGCGGGGC TGTGGCGAGA TTGGCACCCT GCTCCATTGC 4561 TGGTGGGATT GCAAGCTGGT GCAGCCGCTT TGGAAATCAG TCTGGCGCTT TCTGAGGGAC 4621 CTCGAGCTTG AGATTCCCTT CGATCCCGCA ATTCCCTTGC TCGGAATCTA TCCTAACGAA 4681 TACAAGAGCT GTTGTTACAA GGATACGTGT ACCCGGATGT TCATCGCGGC CTTGTTTACG 4741 ATAGCTAAGA CGTGGAATCA GCCTAAGTGC CCCACAATGA TCGATTGGAT CAAGAAAATG 4801 TGGCATATTT ATACCATGGA GTATTACGCA GCAATTAAGA ATGACGAATT TATTTCCTTC 4861 GTTGGGACCT GGATGAAGCT GGAGACTATT ATTCTGAGCA AGCTGTCTCA GGAGCAAAAG 4921 ACAAAGCATA GAATCTTCTC TCTCATTGGT GGTAACGACT ACAAAGACGA TGACGACAAG 4981 TAAAGCGGCC GGGCGCGGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA GGCCGAGGCG 5041 GGAGGATCGC AGTTCGAGAC CAGCGCGAGA CCCCGTCTCT ACAAAAATAC AAAAATTAGC 5101 TTCTAGAAGT TGTCTCCTCC TGCACTGACT GACTGATACA ATCGATTTCT GGATCCGCAG 5161 GCCTAATCAA CCTCTGGATT ACAAAATTTG TGAAAGATTG ACTGGTATTC TTAACTATGT 5221 TGCTCCTTTT ACGCTATGTG GATACGCTGC TTTAATGCCT TTGTATCATG CTATTGCTTC 5281 CCGTATGGCT TTCATTTTCT CCTCCTTGTA TAAATCCTGG TTGCTGTCTC TTTATGAGGA 5341 GTTGTGGCCC GTTGTCAGGC AACGTGGCGT GGTGTGCACT GTGTTTGCTG ACGCAACCCC 5401 CACTGGTTGG GGCATTGCCA CCACCTGTCA GCTCCTTTCC GGGACTTTCG CTTTCCCCCT 5461 CCCTATTGCC ACGGCGGAAC TCATCGCCGC CTGCCTTGCC CGCTGCTGGA CAGGGGCTCG 5521 GCTGTTGGGC ACTGACAATT CCGTGGTGTT GTCGGGGAAG CTGACGTCCT TTCCATGGCT 5581 GCTCGCCTGT GTTGCCACCT GGATTCTGCG CGGGACGTCC TTCTGCTACG TCCCTTCGGC 5641 CCTCAATCCA GCGGACCTTC CTTCCCGCTG AGAGACACAA AAAATTCCAA CACACTATTG 5701 CAATGAAAAT AAATTTCCTT TATTAGCCAG AAGTCAGATG CTCAAGGGGC TTCATGATGT 5761 CCCCATAATT TTTGGCAGAG GGAAAAAGAT CTCAGTGGTA TTTGTGAGCC AGGGCATTGG 5821 CCTTCTGATA GGCAGCCTGC ACCTGAGGAG TGCGGCCGCT TTACTTGTAC AGCTCGTCCA 5881 TGCCGAGAGT GATCCCGGCG GCGGTCACGA ACTCCAGCAG GACCATGTGA TCGCGCTTCT 5941 CGTTGGGGTC TTTGCTCAGG GCGGACTGGG TGCTCAGGTA GTGGTTGTCG GGCAGCAGCA 6001 CGGGGCCGTC GCCGATGGGG GTGTTCTGCT GGTAGTGGTC GGCGAGCTGC ACGCTGCCGT 6061 CCTCGATGTT GTGGCGGATC TTGAAGTTCA CCTTGATGCC GTTCTTCTGC TTGTCGGCCA 6121 TGATATAGAC GTTGTGGCTG TTGTAGTTGT ACTCCAGCTT GTGCCCCAGG ATGTTGCCGT 6181 CCTCCTTGAA GTCGATGCCC TTCAGCTCGA TGCGGTTCAC CAGGGTGTCG CCCTCGAACT 6241 TCACCTCGGC GCGGGTCTTG TAGTTGCCGT CGTCCTTGAA GAAGATGGTG CGCTCCTGGA 6301 CGTAGCCTTC GGGCATGGCG GACTTGAAGA AGTCGTGCTG CTTCATGTGG TCGGGGTAGC 6361 GGCTGAAGCA CTGCACGCCG TAGGTCAGGG TGGTCACGAG GGTGGGCCAG GGCACGGGCA 6421 GCTTGCCGGT GGTGCAGATG AACTTCAGGG TCAGCTTGCC GTAGGTGGCA TCGCCCTCGC 6481 CCTCGCCGGA CACGCTGAAC TTGTGGCCGT TTACGTCGCC GTCCAGCTCG ACCAGGATGG 6541 GCACCACCCC GGTGAACAGC TCCTCGCCCT TGCTCACCAT GGTGGCGGGA TCTGACGGTT 6601 CACTAAACCA GCTCTGCTTA TATAGACCTC CCACCGTACA CGCCTACCGC CCATTTGCGT 6661 CAATGGGGCG GAGTTGTTAC GACATTTTGG AAAGTCCCGT TGATTTTGGT GCCAAAACAA 6721 ACTCCCATTG ACGTCAATGG GGTGGAGACT TGGAAATCCC CGTGAGTCAA ACCGCTATCC 6781 ACGCCCATTG ATGTACTGCC AAAACCGCAT CACCATGGTA ATAGCGATGA CTAATACGTA 6841 GATGTACTGC CAAGTAGGAA AGTCCCATAA GGTCATGTAC TGGGCATAAT GCCAGGCGGG 6901 CCATTTACCG TCATTGACGT CAATAGGGGG CGTACTTGGC ATATGATACA CTTGATGTAC 6961 TGCCAAGTGG GCAGTTTACC GTAAATACTC CACCCATTGA CGTCAATGGA AAGTCCCTAT 7021 TGGCGTTACT ATGGGAACAT ACGTCATTAT TGACGTCAAT GGGCGGGGGT CGTTGGGCGG 7081 TCAGCCAGGC GGGCCATTTA CCGTAAGTTA TGTAACGGGC CTGCTGCCGG CTCTGCGGCC 7141 TCTTCCGCGT CTTCGCCTTC GCCCTCAGAC GAGTCGGATC TCCCTTTGGG CCGCCTCCCC 7201 GCCTGTCTAG CTTGACTGAC TGAGATACAG CGTACCTTCA GCTCACAGAC ATGATAAGAT 7261 ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG AAAAAAATGC TTTATTTGTG 7321 AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG CTGCAATAAA CAAGTT (SEQ ID NO: 43) LINE-1 plasmid CVBE IRES GFP (SEQ ID NO: 44) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGTTA AAACAGCCTG TGGGTTGATC 1081 CCACCCACAG GCCCATTGGG CGCTAGCACT CTGGTATCAC GGTACCTTTG TGCGCCTGTT 1141 TTATACCCCC TCCCCCAACT GTAACTTAGA AGTAACACAC ACCGATCAAC AGTCAGCGTG 1201 GCACACCAGC CACGTTTTGA TCAAGCACTT CTGTTACCCC GGACTGAGTA TCAATAGACT 1261 GCTCACGCGG TTGAAGGAGA AAGCGTTCGT TATCCGGCCA ACTACTTCGA AAAACCTAGT 1321 AACACCGTGG AAGTTGCAGA GTGTTTCGCT CAGCACTACC CCAGTGTAGA TCAGGTCGAT 1381 GAGTCACCGC ATTCCCCACG GGCGACCGTG GCGGTGGCTG CGTTGGCGGC CTGCCCATGG 1441 GGAAACCCAT GGGACGCTCT AATACAGACA TGGTGCGAAG AGTCTATTGA GCTAGTTGGT 1501 AGTCCTCCGG CCCCTGAATG CGGCTAATCC TAACTGCGGA GCACACACCC TCAAGCCAGA 1561 GGGCAGTGTG TCGTAACGGG CAACTCTGCA GCGGAACCGA CTACTTTGGG TGTCCGTGTT 1621 TCATTTTATT CCTATACTGG CTGCTTATGG TGACAATTGA GAGATCGTTA CCATATAGCT 1681 ATTGGATTGG CCATCCGGTG ACTAATAGAG CTATTATATA TCCCTTTGTT GGGTTTATAC 1741 CACTTAGCTT GAAAGAGGTT AAAACATTAC AATTCATTGT TAAGTTGAAT ACAGCAAATA 1801 CATGACCGGC TCTAACTCAC ATATCACCAT CCTTACACTT AACATTAACG GCCTCAACTC 1861 AGCTATCAAG CGCCATCGGC TGGCCAGCTG GATCAAATCA CAGGATCCAA GCGTTTGTTG 1921 CATCCAAGAG ACCCACCTGA CCTGTAGAGA TACTCACCGC CTCAAGATCA AGGGATGGCG 1981 AAAGATTTAT CAGGCGAACG GTAAGCAGAA GAAAGCCGGA GTCGCAATTC TGGTCTCAGA 2041 CAAGACGGAT TTCAAGCCCA CCAAAATTAA GCGTGATAAG GAAGGTCACT ATATTATGGT 2101 GAAAGGCAGC ATACAGCAGG AAGAACTTAC CATATTGAAC ATCTACGCGC CAAACACCGG 2161 CGCACCTCGC TTTATCAAAC AGGTCCTGTC CGATCTGCAG CGAGATCTGG ATTCTCATAC 2221 GTTGATTATG GGTGATTTCA ATACACCATT GAGCACCCTG GATCGCAGCA CCAGGCAAAA 2281 GGTAAATAAA GACACGCAAG AGCTCAATAG CGCACTGCAT CAGGCAGATC TCATTGATAT 2341 TTATCGCACT CTTCATCCTA AGAGTACCGA GTACACATTC TTCAGCGCCC CACATCATAC 2401 ATACTCAAAG ATCGATCATA TCGTCGGCTC AAAGGCTCTG CTGTCAAAGT GCAAGCGCAC 2461 AGAGATAATT ACAAATTACC TGTCAGATCA TAGCGCGATC AAGCTCGAGC TGAGAATCAA 2521 GAACCTGACC CAGAGCCGGA GTACCACTTG GAAGCTTAAT AACCTGCTGC TCAACGATTA 2581 TTGGGTCCAC AATGAGATGA AGGCAGAGAT TAAAATGTTC TTCGAAACAA ATGAGAATAA 2641 GGATACTACC TATCAAAACC TTTGGGATGC CTTTAAGGCC GTCTGCAGAG GCAAGTTCAT 2701 CGCCCTCAAC GCCTATAAAA GAAAACAAGA GAGATCTAAG ATCGATACTC TCACCTCTCA 2761 GCTGAAGGAG TTGGAGAAAC AGGAACAGAC CCACTCCAAG GCGTCAAGAC GGCAGGAGAT 2821 CACAAAGATT CGCGCCGAGT TGAAAGAGAT CGAAACCCAA AAGACTCTTC AGAAAATTAA 2881 CGAGTCTCGT AGTTGGTTCT TCGAGCGGAT TAATAAGATA GACAGACCTC TGGCACGACT 2941 GATTAAGAAG AAGCGCGAAA AGAACCAGAT TGATACCATC AAGAACGACA AGGGCGACAT 3001 CACTACTGAC CCGACCGAGA TCCAGACCAC TATTCGGGAG TATTATAAGC ATTTGTATGC 3061 TAACAAGCTT GAGAACCTGG AAGAGATGGA CACTTTTCTG GATACCTATA CTCTGCCACG 3121 GCTTAATCAA GAGGAAGTCG AGTCCCTCAA CCGCCCAATT ACAGGAAGCG AGATTGTGGC 3181 CATAATTAAC TCCCTGCCGA CAAAGAAATC TCCTGGTCCG GACGGGTTTA CAGCTGAGTT 3241 TTATCAACGG TATATGGAAG AGCTTGTACC GTTTCTGCTC AAGCTCTTTC AGTCTATAGA 3301 AAAGGAAGGC ATCTTGCCCA ATTCCTTCTA CGAAGCTTCT ATAATACTTA TTCCCAAACC 3361 AGGACGCGAT ACCACAAAGA AGGAAAACTT CCGGCCCATT AGTCTCATGA ATATCGACGC 3421 TAAAATATTG AACAAGATTC TCGCCAACAG AATCCAACAA CATATTAAGA AATTGATACA 3481 TCACGACCAG GTGGGGTTTA TACCTGGCAT GCAGGGCTGG TTTAACATCC GGAAGAGTAT 3541 TAACGTCATT CAACACATTA ATAGAGCTAA GGATAAGAAT CATATGATCA TCTCTATAGA 3601 CGCGGAAAAG GCATTCGATA AGATTCAGCA GCCATTTATG CTCAAGACTC TGAACAAACT 3661 CGGCATCGAC GGAACATATT TTAAGATTAT TCGCGCAATT TACGATAAGC CGACTGCTAA 3721 CATTATCCTT AACGGCCAAA AGCTCGAGGC CTTTCCGCTC AAGACTGGAA CCCGCCAAGG 3781 CTGTCCCCTC TCCCCGCTTT TGTTTAATAT TGTACTCGAG GTGCTGGCTA GGGCTATTCG 3841 TCAAGAGAAA GAGATTAAAG GGATACAGCT CGGGAAGGAA GAGGTCAAGC TTTCCTTGTT 3901 CGCCGATGAT ATGATTGTGT ACCTGGAGAA TCCTATTGTG TCTGCTCAGA ACCTTCTTAA 3961 ACTTATTTCT AACTTTAGCA AGGTCAGCGG CTATAAGATT AACGTCCAGA AATCTCAGGC 4021 CTTTCTGTAC ACAAATAATC GACAGACCGA ATCCCAGATA ATGGGTGAGC TTCCGTTTGT 4081 CATAGCCAGC AAAAGGATAA AGTATCTCGG AATCCAGCTG ACACGAGACG TTAAAGATTT 4141 GTTTAAGGAA AATTACAAGC CTCTCCTGAA AGAGATTAAG GAAGATACTA ATAAGTGGAA 4201 GAATATCCCC TGTTCATGGG TTGGCAGAAT CAACATAGTG AAGATGGCAA TACTTCCTAA 4261 AGTGATATAT CGCTTTAACG CCATCCCAAT TAAACTGCCT ATGACCTTCT TTACGGAGCT 4321 CGAGAAAACA ACCCTTAAAT TTATATGGAA TCAAAAGAGA GCAAGAATAG CGAAGTCCAT 4381 CTTGAGCCAG AAGAATAAGG CCGGTGGGAT TACTTTGCCT GATTTTAAGT TGTATTATAA 4441 AGCCACAGTA ACTAAGACAG CCTGGTATTG GTATCAGAAT AGAGACATCG ACCAGTGGAA 4501 TCGGACCGAA CCATCAGAGA TAATGCCCCA CATCTATAAT TACCTTATAT TCGATAAGCC 4561 AGAAAAGAAT AAACAGTGGG GCAAAGACAG CCTCTTCAAC AAGTGGTGTT GGGAGAATTG 4621 GCTGGCCATA TGCCGGAAAC TCAAGCTCGA CCCCTTTCTT ACACCCTACA CTAAAATCAA 4681 CAGTAGGTGG ATCAAGGACT TGAATGTCAA GCCAAAGACT ATAAAGACAC TGGAAGAGAA 4741 TCTTGGGATC ACAATACAAG ATATAGGCGT CGGCAAAGAT TTTATGTCAA AGACGCCCAA 4801 GGCCATGGCC ACTAAGGATA AGATTGATAA GTGGGACCTT ATTAAGCTCA AAAGCTTCTG 4861 TACTGCCAAG GAGACCACGA TCAGAGTTAA TAGGCAGCCC ACTACATGGG AAAAGATTTT 4921 CGCCACTTAT TCATCAGATA AGGGGTTGAT AAGCAGAATA TATAACGAGC TGAAGCAGAT 4981 CTACAAGAAG AAAACGAATA ATCCCATCAA GAAGTGGGCA AAAGATATGA ACAGGCATTT 5041 TAGCAAAGAG GATATCTACG CCGCGAAGAA GCATATGAAG AAGTGTAGTT CAAGCTTGGC 5101 CATTCGTGAG ATGCAGATTA AGACGACCAT GCGATACCAC CTTACCCCAG TGAGGATGGC 5161 AATTATCAAG AAATCTGGCA ATAATAGATG TTGGCGGGGC TGTGGCGAGA TTGGCACCCT 5221 GCTCCATTGC TGGTGGGATT GCAAGCTGGT GCAGCCGCTT TGGAAATCAG TCTGGCGCTT 5281 TCTGAGGGAC CTCGAGCTTG AGATTCCCTT CGATCCCGCA ATTCCCTTGC TCGGAATCTA 5341 TCCTAACGAA TACAAGAGCT GTTGTTACAA GGATACGTGT ACCCGGATGT TCATCGCGGC 5401 CTTGTTTACG ATAGCTAAGA CGTGGAATCA GCCTAAGTGC CCCACAATGA TCGATTGGAT 5461 CAAGAAAATG TGGCATATTT ATACCATGGA GTATTACGCA GCAATTAAGA ATGACGAATT 5521 TATTTCCTTC GTTGGGACCT GGATGAAGCT GGAGACTATT ATTCTGAGCA AGCTGTCTCA 5581 GGAGCAAAAG ACAAAGCATA GAATCTTCTC TCTCATTGGT GGTAACGACT ACAAAGACGA 5641 TGACGACAAG TAAAGCGCTT CTAGAAGTTG TCTCCTCCTG CACTGACTGA CTGATACAAT 5701 CGATTTCTGG ATCCGCAGGC CTAATCAACC TCTGGATTAC AAAATTTGTG AAAGATTGAC 5761 TGGTATTCTT AACTATGTTG CTCCTTTTAC GCTATGTGGA TACGCTGCTT TAATGCCTTT 5821 GTATCATGCT ATTGCTTCCC GTATGGCTTT CATTTTCTCC TCCTTGTATA AATCCTGGTT 5881 GCTGTCTCTT TATGAGGAGT TGTGGCCCGT TGTCAGGCAA CGTGGCGTGG TGTGCACTGT 5941 GTTTGCTGAC GCAACCCCCA CTGGTTGGGG CATTGCCACC ACCTGTCAGC TCCTTTCCGG 6001 GACTTTCGCT TTCCCCCTCC CTATTGCCAC GGCGGAACTC ATCGCCGCCT GCCTTGCCCG 6061 CTGCTGGACA GGGGCTCGGC TGTTGGGCAC TGACAATTCC GTGGTGTTGT CGGGGAAGCT 6121 GACGTCCTTT CCATGGCTGC TCGCCTGTGT TGCCACCTGG ATTCTGCGCG GGACGTCCTT 6181 CTGCTACGTC CCTTCGGCCC TCAATCCAGC GGACCTTCCT TCCCGCGAAC AAACGACCCA 6241 ACACCCGTGC GTTTTATTCT GTCTTTTTAT TGCCGATCCC CTCAGAAGAA CTCGTCAAGA 6301 AGGCGATAGA AGGCGATGCG CTGCGAATCG GGAGCGGCGA TACCGTAAAG CACGAGGAAG 6361 CGGTCAGCCC ATTCGCCGCC AAGCTCTTCA GCAATATCAC GGGTAGCCAA CGCTATGTCC 6421 TGATAGCGGT CGGCCGCTTT ACTTGTACAG CTCGTCCATG CCGAGAGTGA TCCCGGCGGC 6481 GGTCACGAAC TCCAGCAGGA CCATGTGATC GCGCTTCTCG TTGGGGTCTT TGCTCAGGGC 6541 GGACTGGGTG CTCAGGTAGT GGTTGTCGGG CAGCAGCACG GGGCCGTCGC CGATGGGGGT 6601 GTTCTGCTGG TAGTGGTCGG CCAGGTGAGT CCAGGAGATG TTTCAGCACT GTTGCCTTTA 6661 GTCTCGAGGC AACTTAGACA ACTGAGTATT GATCTGAGCA CAGCAGGGTG TGAGCTGTTT 6721 GAAGATACTG GGGTTGGGGG TGAAGAAACT GCAGAGGACT AACTGGGCTG AGACCCAGTG 6781 GCAATGTTTT AGGGCCTAAG GAATGCCTCT GAAAATCTAG ATGGACAACT TTGACTTTGA 6841 GAAAAGAGAG GTGGAAATGA GGAAAATGAC TTTTCTTTAT TAGATTTCGG TAGAAAGAAC 6901 TTTCATCTTT CCCCTATTTT TGTTATTCGT TTTAAAACAT CTATCTGGAG GCAGGACAAG 6961 TATGGTCATT AAAAAGATGC AGGCAGAAGG CATATATTGG CTCAGTCAAA GTGGGGAACT 7021 TTGGTGGCCA AACATACATT GCTAAGGCTA TTCCTATATC AGCTGGACAC ATATAAAATG 7081 CTGCTAATGC TTCATTACAA ACTTATATCC TTTAATTCCA GATGGGGGCA AAGTATGTCC 7141 AGGGGTGAGG AACAATTGAA ACATTTGGGC TGGAGTAGAT TTTGAAAGTC AGCTCTGTGT 7201 GTGTGTGTGT GTGTGTGTGT GTGAGAGCGT GTGTTTCTTT TAACGTTTTC AGCCTACAGC 7261 ATACAGGGTT CATGGTGGCA AGAAGATAAC AAGATTTAAA TTATGGCCAG TGACTAGTGC 7321 TGCAAGAAGA ACAACTACCT GCATTTAATG GGAAAGCAAA ATCTCAGGCT TTGAGGGAAG 7381 TTAACATAGG CTTGATTCTG GGTGGAAGCT GGGTGTGTAG TTATCTGGAG GCCAGGCTGG 7441 AGCTCTCAGC TCACTATGGG TTCATCTTTA TTGTCTCCTT TCATCTCAAC AGCTGCACGC 7501 TGCCGTCCTC GATGTTGTGG CGGATCTTGA AGTTCACCTT GATGCCGTTC TTCTGCTTGT 7561 CGGCCATGAT ATAGACGTTG TGGCTGTTGT AGTTGTACTC CAGCTTGTGC CCCAGGATGT 7621 TGCCGTCCTC CTTGAAGTCG ATGCCCTTCA GCTCGATGCG GTTCACCAGG GTGTCGCCCT 7681 CGAACTTCAC CTCGGCGCGG GTCTTGTAGT TGCCGTCGTC CTTGAAGAAG ATGGTGCGCT 7741 CCTGGACGTA GCCTTCGGGC ATGGCGGACT TGAAGAAGTC GTGCTGCTTC ATGTGGTCGG 7801 GGTAGCGGCT GAAGCACTGC ACGCCGTAGG TCAGGGTGGT CACGAGGGTG GGCCAGGGCA 7861 CGGGCAGCTT GCCGGTGGTG CAGATGAACT TCAGGGTCAG CTTGCCGTAG GTGGCATCGC 7921 CCTCGCCCTC GCCGGACACG CTGAACTTGT GGCCGTTTAC GTCGCCGTCC AGCTCGACCA 7981 GGATGGGCAC CACCCCGGTG AACAGCTCCT CGCCCTTGCT CACCATGGTG GCGAATTCGA 8041 AGCTTGAGCA CGAGATCTGA GTCCGGTAGG CCTAGCGGAT CTGACGGTTC ACTAAACCAG 8101 CTCTGCTTAT ATAGACCTCC CACCGTACAC GCCTACCGCC CATTTGCGTC AATGGGGCGG 8161 AGTTGTTACG ACATTTTGGA AAGTCCCGTT GATTTTGGTG CCAAAACAAA CTCCCATTGA 8221 CGTCAATGGG GTGGAGACTT GGAAATCCCC GTGAGTCAAA CCGCTATCCA CGCCCATTGA 8281 TGTACTGCCA AAACCGCATC ACCATGGTAA TAGCGATGAC TAATACGTAG ATGTACTGCC 8341 AAGTAGGAAA GTCCCATAAG GTCATGTACT GGGCATAATG CCAGGCGGGC CATTTACCGT 8401 CATTGACGTC AATAGGGGGC GTACTTGGCA TATGATACAC TTGATGTACT GCCAAGTGGG 8461 CAGTTTACCG TAAATACTCC ACCCATTGAC GTCAATGGAA AGTCCCTATT GGCGTTACTA 8521 TGGGAACATA CGTCATTATT GACGTCAATG GGCGGGGGTC GTTGGGCGGT CAGCCAGGCG 8581 GGCCATTTAC CGTAAGTTAT GTAACGGGCC TGCTGCCGGC TCTGCGGCCT CTTCCGCGTC 8641 TTCGCCTTCG CCCTCAGACG AGTCGGATCT CCCTTTGGGC CGCCTCCCCG CCTGTCTAGC 8701 TTGACTGACT GAGATACAGC GTACCTTCAG CTCACAGACA TGATAAGATA CATTGATGAG 8761 TTTGGACAAA CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA AATTTGTGAT 8821 GCTATTGCTT TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA CAACAATTGC 8881 ATTCATTTTA TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG CAAGTAAAAC 8941 CTCTACAAAT GTGGTATTGG CCCATCTCTA TCGGTATCGT AGCATAACCC CTTGGGGCCT 9001 CTAAACGGGT CTTGAGGGGT TTTTTGTGCC CCTCGGGCCG GATTGCTATC TACCGGCATT 9061 GGCGCAGAAA AAAATGCCTG ATGCGACGCT GCGCGTCTTA TACTCCCACA TATGCCAGAT 9121 TCAGCAACGG ATACGGCTTC CCCAACTTGC CCACTTCCAT ACGTGTCCTC CTTACCAGAA 9181 ATTTATCCTT AAGGTCGTCA GCTATCCTGC AGGCGATCTC TCGATTTCGA TCAAGACATT 9241 CCTTTAATGG TCTTTTCTGG ACACCACTAG GGGTCAGAAG TAGTTCATCA AACTTTCTTC 9301 CCTCCCTAAT CTCATTGGTT ACCTTGGGCT ATCGAAACTT AATTAAGCGA TCTGCATCTC 9361 AATTAGTCAG CAACCATAGT CCCGCCCCTA ACTCCGCCCA TCCCGCCCCT AACTCCGCCC 9421 AGTTCCGCCC ATTCTCCGCC CCATCGCTGA CTAATTTTTT TTATTTATGC AGAGGCCGAG 9481 GCCGCCTCGG CCTCTGAGCT ATTCCAGAAG TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC 9541 TTTTGCAAAG GAGGTAGCCA ACATGATTGA ACAAGATGGA TTGCACGCAG GTTCTCCCGC 9601 CGCTTGGGTG GAGAGGCTAT TCGGCTATGA CTGGGCACAA CAGACAATCG GCTGCTCTGA 9661 TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG GCGCCCGGTT CTTTTTGTCA AGACCGACCT 9721 GTCCGGTGCC CTGAATGAAC TCCAGGACGA GGCAGCGCGG CTATCGTGGC TGGCCACGAC 9781 GGGCGTTCCT TGCGCAGCTG TGCTCGACGT TGTCACTGAA GCGGGAAGGG ACTGGCTGCT 9841 ATTGGGCGAA GTGCCGGGGC AGGATCTCCT GTCATCTCAC CTTGCTCCTG CCGAGAAAGT 9901 ATCCATCATG GCTGATGCAA TGCGGCGGCT GCATACGCTT GATCCGGCTA CCTGCCCATT 9961 CGACCACCAA GCGAAACATC GCATCGAGCG AGCACGTACT CGGATGGAAG CCGGTCTTGT 10021 CGATCAGGAT GATCTGGACG AAGAGCATCA GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG 10081 GCTCAAGGCG CGGATGCCCG ACGGCGAGGA TCTCGTCGTG ACCCACGGCG ATGCCTGCTT 10141 GCCGAATATC ATGGTGGAAA ATGGCCGCTT TTCTGGATTC ATCGACTGTG GCCGGCTGGG 10201 TGTGGCGGAC CGCTATCAGG ACATAGCGTT GGCTACCCGT GATATTGCTG AAGAGCTTGG 10261 CGGCGAATGG GCTGACCGCT TCCTCGTGCT TTACGGTATC GCCGCTCCCG ATTCGCAGCG 10321 CATCGCCTTC TATCGCCTTC TTGACGAGTT CTTCTAGTAT GTAAGCCCTG TGCCTTCTAG 10381 TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC 10441 TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA 10501 TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG 10561 CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGTTAATTAA CCAGTCAAGT CAGCTACTTG 10621 GCGAGATCGA CTTGTCTGGG TTTCGACTAC GCTCAGAATT GCGTCAGTCA AGTTCGATCT 10681 GGTCCTTGCT ATTGCACCCG TTCTCCGATT ACGAGTTTCA TTTAAATCAT GTGAGCAAAA 10741 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC 10801 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA 10861 GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG 10921 ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 10981 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT 11041 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG 11101 TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC 11161 AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC 11221 ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 11281 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC 11341 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG 11401 GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA 11461 AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT 11521 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 11581 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCATTTAAAT TTCCGAACTC TCCAAGGCCC 11641 TCGTCGGAAA ATCTTCAAAC CTTTCGTCCG ATCCATCTTG CAGGCTACCT CTCGAACGAA 11701 CTATCGCAAG TCTCTTGGCC GGCCTTGCGC CTTGGCTATT GCTTGGCAGC GCCTATCGCC 11761 AGGTATTACT CCAATCCCGA ATATCCGAGA TCGGGATCAC CCGAGAGAAG TTCAACCTAC 11821 ATCCTCAATC CCGATCTATC CGAGATCCGA GGAATATCGA AATCGGGGCG CGCCTGGTGT 11881 ACCGAGAACG ATCCTCTCAG TGCGAGTCTC GACGATCCAT ATCGTTGCTT GGCAGTCAGC 11941 CAGTCGGAAT CCAGCTTGGG ACCCAGGAAG TCCAATCGTC AGATATTGTA CTCAAGCCTG 12001 GTCACGGCAG CGTACCGATC TGTTTAAACC TAGATATTGA TAGTCTGATC GGTCAACGTA 12061 TAATCGAGTC CTAGCTTTTG CAAACATCTA TCAAGAGACA GGATCAGCAG GAGGCTTTCG 12121 CATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC 12181 TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC 12241 GCGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC 12301 CGAAGAACGC TTTCCAATGA TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC 12361 CCGTATTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT 12421 GGTTGAGTAT TCACCAGTCA CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT 12481 ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT 12541 TGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG GGGGATCATG TAACTCGCCT 12601 TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT 12661 GCCTGTAGCA ATGGCAACAA CCTTGCGTAA ACTATTAACT GGCGAACTAC TTACTCTAGC 12721 TTCCCGGCAA CAGTTGATAG ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG 12781 CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC 12841 TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA 12901 CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC 12961 CTCACTGATT AAGCATTGGT AACCGATTCT AGGTGCATTG GCGCAGAAAA AAATGCCTGA 13021 TGCGACGCTG CGCGTCTTAT ACTCCCACAT ATGCCAGATT CAGCAACGGA TACGGCTTCC 13081 CCAACTTGCC CACTTCCATA CGTGTCCTCC TTACCAGAAA TTTATCCTTA AGATCGTTTA 13141 AACTCGACTC TGGCTCTATC GAATCTCCGT CGTTTCGAGC TTACGCGAAC AGCCGTGGCG 13201 CTCATTTGCT CGTCGGGCAT CGAATCTCGT CAGCTATCGT CAGCTTACCT TTTTGGCAGC 13261 GATCGCGGCT CCCGACATCT TGGACCATTA GCTCCACAGG TATCTTCTTC CCTCTAGTGG 13321 TCATAACAGC AGCTTCAGCT ACCTCTCAAT TCAAAAAACC CCTCAAGACC CGTTTAGAGG 13381 CCCCAAGGGG TTATGCTATC AATCGTTGCG TTACACACAC AAAAAACCAA CACACATCCA 13441 TCTTCGATGG ATAGCGATTT TATTATCTAA CTGCTGATCG AGTGTAGCCA GATCTAGTAA 13501 TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG 13561 GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG 13621 TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA 13681 CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT 13741 GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC 13801 TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGCT GATGCGGTTT 13861 TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC 13921 CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT 13981 CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT 14041 ATAAGCAGAG CTGGTTTAGT GAACCGTCAG ATCAGATCTT TGTCGATCCT ACCATCCACT 14101 CGACACACCC GCCAGCGGCC GC (SEQ ID NO: 44) LINE-1 Plasmid EV71 IRES (SEQ ID NO: 45) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGTTA AAACAGCTGT GGGTTGTCAC 1081 CCACCCACAG GGTCCACTGG GCGCTAGTAC ACTGGTATCT CGGTACCTTT GTACGCCTGT 1141 TTTATACCCC CTCCCTGATT TGCAACTTAG AAGCAACGCA AACCAGATCA ATAGTAGGTG 1201 TGACATACCA GTCGCATCTT GATCAAGCAC TTCTGTATCC CCGGACCGAG TATCAATAGA 1261 CTGTGCACAC GGTTGAAGGA GAAAACGTCC GTTACCCGGC TAACTACTTC GAGAAGCCTA 1321 GTAACGCCAT TGAAGTTGCA GAGTGTTTCG CTCAGCACTC CCCCCGTGTA GATCAGGTCG 1381 ATGAGTCACC GCATTCCCCA CGGGCGACCG TGGCGGTGGC TGCGTTGGCG GCCTGCCTAT 1441 GGGGTAACCC ATAGGACGCT CTAATACGGA CATGGCGTGA AGAGTCTATT GAGCTAGTTA 1501 GTAGTCCTCC GGCCCCTGAA TGCGGCTAAT CCTAACTGCG GAGCACATAC CCTTAATCCA 1561 AAGGGCAGTG TGTCGTAACG GGCAACTCTG CAGCGGAACC GACTACTTTG GGTGTCCGTG 1621 TTTCTTTTTA TTCTTGTATT GGCTGCTTAT GGTGACAATT AAAGAATTGT TACCATATAG 1681 CTATTGGATT GGCCATCCAG TGTCAAACAG AGCTATTGTA TATCTCTTTG TTGGATTCAC 1741 ACCTCTCACT CTTGAAACGT TACACACCCT CAATTACATT ATACTGCTGA ACACGAAGCG 1801 TACATGACCG GCTCTAACTC ACATATCACC ATCCTTACAC TTAACATTAA CGGCCTCAAC 1861 TCAGCTATCA AGCGCCATCG GCTGGCCAGC TGGATCAAAT CACAGGATCC AAGCGTTTGT 1921 TGCATCCAAG AGACCCACCT GACCTGTAGA GATACTCACC GCCTCAAGAT CAAGGGATGG 1981 CGAAAGATTT ATCAGGCGAA CGGTAAGCAG AAGAAAGCCG GAGTCGCAAT TCTGGTCTCA 2041 GACAAGACGG ATTTCAAGCC CACCAAAATT AAGCGTGATA AGGAAGGTCA CTATATTATG 2101 GTGAAAGGCA GCATACAGCA GGAAGAACTT ACCATATTGA ACATCTACGC GCCAAACACC 2161 GGCGCACCTC GCTTTATCAA ACAGGTCCTG TCCGATCTGC AGCGAGATCT GGATTCTCAT 2221 ACGTTGATTA TGGGTGATTT CAATACACCA TTGAGCACCC TGGATCGCAG CACCAGGCAA 2281 AAGGTAAATA AAGACACGCA AGAGCTCAAT AGCGCACTGC ATCAGGCAGA TCTCATTGAT 2341 ATTTATCGCA CTCTTCATCC TAAGAGTACC GAGTACACAT TCTTCAGCGC CCCACATCAT 2401 ACATACTCAA AGATCGATCA TATCGTCGGC TCAAAGGCTC TGCTGTCAAA GTGCAAGCGC 2461 ACAGAGATAA TTACAAATTA CCTGTCAGAT CATAGCGCGA TCAAGCTCGA GCTGAGAATC 2521 AAGAACCTGA CCCAGAGCCG GAGTACCACT TGGAAGCTTA ATAACCTGCT GCTCAACGAT 2581 TATTGGGTCC ACAATGAGAT GAAGGCAGAG ATTAAAATGT TCTTCGAAAC AAATGAGAAT 2641 AAGGATACTA CCTATCAAAA CCTTTGGGAT GCCTTTAAGG CCGTCTGCAG AGGCAAGTTC 2701 ATCGCCCTCA ACGCCTATAA AAGAAAACAA GAGAGATCTA AGATCGATAC TCTCACCTCT 2761 CAGCTGAAGG AGTTGGAGAA ACAGGAACAG ACCCACTCCA AGGCGTCAAG ACGGCAGGAG 2821 ATCACAAAGA TTCGCGCCGA GTTGAAAGAG ATCGAAACCC AAAAGACTCT TCAGAAAATT 2881 AACGAGTCTC GTAGTTGGTT CTTCGAGCGG ATTAATAAGA TAGACAGACC TCTGGCACGA 2941 CTGATTAAGA AGAAGCGCGA AAAGAACCAG ATTGATACCA TCAAGAACGA CAAGGGCGAC 3001 ATCACTACTG ACCCGACCGA GATCCAGACC ACTATTCGGG AGTATTATAA GCATTTGTAT 3061 GCTAACAAGC TTGAGAACCT GGAAGAGATG GACACTTTTC TGGATACCTA TACTCTGCCA 3121 CGGCTTAATC AAGAGGAAGT CGAGTCCCTC AACCGCCCAA TTACAGGAAG CGAGATTGTG 3181 GCCATAATTA ACTCCCTGCC GACAAAGAAA TCTCCTGGTC CGGACGGGTT TACAGCTGAG 3241 TTTTATCAAC GGTATATGGA AGAGCTTGTA CCGTTTCTGC TCAAGCTCTT TCAGTCTATA 3301 GAAAAGGAAG GCATCTTGCC CAATTCCTTC TACGAAGCTT CTATAATACT TATTCCCAAA 3361 CCAGGACGCG ATACCACAAA GAAGGAAAAC TTCCGGCCCA TTAGTCTCAT GAATATCGAC 3421 GCTAAAATAT TGAACAAGAT TCTCGCCAAC AGAATCCAAC AACATATTAA GAAATTGATA 3481 CATCACGACC AGGTGGGGTT TATACCTGGC ATGCAGGGCT GGTTTAACAT CCGGAAGAGT 3541 ATTAACGTCA TTCAACACAT TAATAGAGCT AAGGATAAGA ATCATATGAT CATCTCTATA 3601 GACGCGGAAA AGGCATTCGA TAAGATTCAG CAGCCATTTA TGCTCAAGAC TCTGAACAAA 3661 CTCGGCATCG ACGGAACATA TTTTAAGATT ATTCGCGCAA TTTACGATAA GCCGACTGCT 3721 AACATTATCC TTAACGGCCA AAAGCTCGAG GCCTTTCCGC TCAAGACTGG AACCCGCCAA 3781 GGCTGTCCCC TCTCCCCGCT TTTGTTTAAT ATTGTACTCG AGGTGCTGGC TAGGGCTATT 3841 CGTCAAGAGA AAGAGATTAA AGGGATACAG CTCGGGAAGG AAGAGGTCAA GCTTTCCTTG 3901 TTCGCCGATG ATATGATTGT GTACCTGGAG AATCCTATTG TGTCTGCTCA GAACCTTCTT 3961 AAACTTATTT CTAACTTTAG CAAGGTCAGC GGCTATAAGA TTAACGTCCA GAAATCTCAG 4021 GCCTTTCTGT ACACAAATAA TCGACAGACC GAATCCCAGA TAATGGGTGA GCTTCCGTTT 4081 GTCATAGCCA GCAAAAGGAT AAAGTATCTC GGAATCCAGC TGACACGAGA CGTTAAAGAT 4141 TTGTTTAAGG AAAATTACAA GCCTCTCCTG AAAGAGATTA AGGAAGATAC TAATAAGTGG 4201 AAGAATATCC CCTGTTCATG GGTTGGCAGA ATCAACATAG TGAAGATGGC AATACTTCCT 4261 AAAGTGATAT ATCGCTTTAA CGCCATCCCA ATTAAACTGC CTATGACCTT CTTTACGGAG 4321 CTCGAGAAAA CAACCCTTAA ATTTATATGG AATCAAAAGA GAGCAAGAAT AGCGAAGTCC 4381 ATCTTGAGCC AGAAGAATAA GGCCGGTGGG ATTACTTTGC CTGATTTTAA GTTGTATTAT 4441 AAAGCCACAG TAACTAAGAC AGCCTGGTAT TGGTATCAGA ATAGAGACAT CGACCAGTGG 4501 AATCGGACCG AACCATCAGA GATAATGCCC CACATCTATA ATTACCTTAT ATTCGATAAG 4561 CCAGAAAAGA ATAAACAGTG GGGCAAAGAC AGCCTCTTCA ACAAGTGGTG TTGGGAGAAT 4621 TGGCTGGCCA TATGCCGGAA ACTCAAGCTC GACCCCTTTC TTACACCCTA CACTAAAATC 4681 AACAGTAGGT GGATCAAGGA CTTGAATGTC AAGCCAAAGA CTATAAAGAC ACTGGAAGAG 4741 AATCTTGGGA TCACAATACA AGATATAGGC GTCGGCAAAGATTTTATGTC AAAGACGCCC 4801 AAGGCCATGG CCACTAAGGA TAAGATTGAT AAGTGGGACC TTATTAAGCT CAAAAGCTTC 4861 TGTACTGCCA AGGAGACCAC GATCAGAGTT AATAGGCAGC CCACTACATG GGAAAAGATT 4921 TTCGCCACTT ATTCATCAGA TAAGGGGTTG ATAAGCAGAA TATATAACGA GCTGAAGCAG 4981 ATCTACAAGA AGAAAACGAA TAATCCCATC AAGAAGTGGG CAAAAGATAT GAACAGGCAT 5041 TTTAGCAAAG AGGATATCTA CGCCGCGAAG AAGCATATGA AGAAGTGTAG TTCAAGCTTG 5101 GCCATTCGTG AGATGCAGAT TAAGACGACC ATGCGATACC ACCTTACCCC AGTGAGGATG 5161 GCAATTATCA AGAAATCTGG CAATAATAGA TGTTGGCGGG GCTGTGGCGA GATTGGCACC 5221 CTGCTCCATT GCTGGTGGGA TTGCAAGCTG GTGCAGCCGC TTTGGAAATC AGTCTGGCGC 5281 TTTCTGAGGG ACCTCGAGCT TGAGATTCCC TTCGATCCCG CAATTCCCTT GCTCGGAATC 5341 TATCCTAACG AATACAAGAG CTGTTGTTAC AAGGATACGT GTACCCGGAT GTTCATCGCG 5401 GCCTTGTTTA CGATAGCTAA GACGTGGAAT CAGCCTAAGT GCCCCACAAT GATCGATTGG 5461 ATCAAGAAAA TGTGGCATAT TTATACCATG GAGTATTACG CAGCAATTAA GAATGACGAA 5521 TTTATTTCCT TCGTTGGGAC CTGGATGAAG CTGGAGACTA TTATTCTGAG CAAGCTGTCT 5581 CAGGAGCAAA AGACAAAGCA TAGAATCTTC TCTCTCATTG GTGGTAACGA CTACAAAGAC 5641 GATGACGACA AGTAAAGCGC TTCTAGAAGT TGTCTCCTCC TGCACTGACT GACTGATACA 5701 ATCGATTTCT GGATCCGCAG GCCTAATCAA CCTCTGGATT ACAAAATTTG TGAAAGATTG 5761 ACTGGTATTC TTAACTATGT TGCTCCTTTT ACGCTATGTG GATACGCTGC TTTAATGCCT 5821 TTGTATCATG CTATTGCTTC CCGTATGGCT TTCATTTTCT CCTCCTTGTA TAAATCCTGG 5881 TTGCTGTCTC TTTATGAGGA GTTGTGGCCC GTTGTCAGGC AACGTGGCGT GGTGTGCACT 5941 GTGTTTGCTG ACGCAACCCC CACTGGTTGG GGCATTGCCA CCACCTGTCA GCTCCTTTCC 6001 GGGACTTTCG CTTTCCCCCT CCCTATTGCC ACGGCGGAAC TCATCGCCGC CTGCCTTGCC 6061 CGCTGCTGGA CAGGGGCTCG GCTGTTGGGC ACTGACAATT CCGTGGTGTT GTCGGGGAAG 6121 CTGACGTCCT TTCCATGGCT GCTCGCCTGT GTTGCCACCT GGATTCTGCG CGGGACGTCC 6181 TTCTGCTACG TCCCTTCGGC CCTCAATCCA GCGGACCTTC CTTCCCGCGA ACAAACGACC 6241 CAACACCCGT GCGTTTTATT CTGTCTTTTT ATTGCCGATC CCCTCAGAAG AACTCGTCAA 6301 GAAGGCGATA GAAGGCGATG CGCTGCGAAT CGGGAGCGGC GATACCGTAA AGCACGAGGA 6361 AGCGGTCAGC CCATTCGCCG CCAAGCTCTT CAGCAATATC ACGGGTAGCC AACGCTATGT 6421 CCTGATAGCG GTCGGCCGCT TTACTTGTAC AGCTCGTCCA TGCCGAGAGT GATCCCGGCG 6481 GCGGTCACGA ACTCCAGCAG GACCATGTGA TCGCGCTTCT CGTTGGGGTC TTTGCTCAGG 6541 GCGGACTGGG TGCTCAGGTA GTGGTTGTCG GGCAGCAGCA CGGGGCCGTC GCCGATGGGG 6601 GTGTTCTGCT GGTAGTGGTC GGCCAGGTGA GTCCAGGAGA TGTTTCAGCA CTGTTGCCTT 6661 TAGTCTCGAG GCAACTTAGA CAACTGAGTA TTGATCTGAG CACAGCAGGG TGTGAGCTGT 6721 TTGAAGATAC TGGGGTTGGG GGTGAAGAAA CTGCAGAGGA CTAACTGGGC TGAGACCCAG 6781 TGGCAATGTT TTAGGGCCTA AGGAATGCCT CTGAAAATCT AGATGGACAA CTTTGACTTT 6841 GAGAAAAGAG AGGTGGAAAT GAGGAAAATG ACTTTTCTTT ATTAGATTTC GGTAGAAAGA 6901 ACTTTCATCT TTCCCCTATT TTTGTTATTC GTTTTAAAAC ATCTATCTGG AGGCAGGACA 6961 AGTATGGTCA TTAAAAAGAT GCAGGCAGAA GGCATATATT GGCTCAGTCA AAGTGGGGAA 7021 CTTTGGTGGC CAAACATACA TTGCTAAGGC TATTCCTATA TCAGCTGGAC ACATATAAAA 7081 TGCTGCTAAT GCTTCATTAC AAACTTATAT CCTTTAATTC CAGATGGGGG CAAAGTATGT 7141 CCAGGGGTGA GGAACAATTG AAACATTTGG GCTGGAGTAG ATTTTGAAAG TCAGCTCTGT 7201 GTGTGTGTGT GTGTGTGTGT GTGTGAGAGC GTGTGTTTCT TTTAACGTTT TCAGCCTACA 7261 GCATACAGGG TTCATGGTGG CAAGAAGATA ACAAGATTTA AATTATGGCC AGTGACTAGT 7321 GCTGCAAGAA GAACAACTAC CTGCATTTAA TGGGAAAGCA AAATCTCAGG CTTTGAGGGA 7381 AGTTAACATA GGCTTGATTC TGGGTGGAAG CTGGGTGTGT AGTTATCTGG AGGCCAGGCT 7441 GGAGCTCTCA GCTCACTATG GGTTCATCTT TATTGTCTCC TTTCATCTCA ACAGCTGCAC 7501 GCTGCCGTCC TCGATGTTGT GGCGGATCTT GAAGTTCACC TTGATGCCGT TCTTCTGCTT 7561 GTCGGCCATG ATATAGACGT TGTGGCTGTT GTAGTTGTAC TCCAGCTTGT GCCCCAGGAT 7621 GTTGCCGTCC TCCTTGAAGT CGATGCCCTT CAGCTCGATG CGGTTCACCA GGGTGTCGCC 7681 CTCGAACTTC ACCTCGGCGC GGGTCTTGTA GTTGCCGTCG TCCTTGAAGA AGATGGTGCG 7741 CTCCTGGACG TAGCCTTCGG GCATGGCGGA CTTGAAGAAG TCGTGCTGCT TCATGTGGTC 7801 GGGGTAGCGG CTGAAGCACT GCACGCCGTA GGTCAGGGTG GTCACGAGGG TGGGCCAGGG 7861 CACGGGCAGC TTGCCGGTGG TGCAGATGAA CTTCAGGGTC AGCTTGCCGT AGGTGGCATC 7921 GCCCTCGCCC TCGCCGGACA CGCTGAACTT GTGGCCGTTT ACGTCGCCGT CCAGCTCGAC 7981 CAGGATGGGC ACCACCCCGG TGAACAGCTC CTCGCCCTTG CTCACCATGG TGGCGAATTC 8041 GAAGCTTGAG CACGAGATCT GAGTCCGGTA GGCCTAGCGG ATCTGACGGT TCACTAAACC 8101 AGCTCTGCTT ATATAGACCT CCCACCGTAC ACGCCTACCG CCCATTTGCG TCAATGGGGC 8161 GGAGTTGTTA CGACATTTTG GAAAGTCCCG TTGATTTTGG TGCCAAAACA AACTCCCATT 8221 GACGTCAATG GGGTGGAGAC TTGGAAATCC CCGTGAGTCA AACCGCTATC CACGCCCATT 8281 GATGTACTGC CAAAACCGCA TCACCATGGT AATAGCGATG ACTAATACGT AGATGTACTG 8341 CCAAGTAGGA AAGTCCCATA AGGTCATGTA CTGGGCATAA TGCCAGGCGG GCCATTTACC 8401 GTCATTGACG TCAATAGGGG GCGTACTTGG CATATGATAC ACTTGATGTA CTGCCAAGTG 8461 GGCAGTTTAC CGTAAATACT CCACCCATTG ACGTCAATGG AAAGTCCCTA TTGGCGTTAC 8521 TATGGGAACA TACGTCATTA TTGACGTCAA TGGGCGGGGG TCGTTGGGCG GTCAGCCAGG 8581 CGGGCCATTT ACCGTAAGTT ATGTAACGGG CCTGCTGCCG GCTCTGCGGC CTCTTCCGCG 8641 TCTTCGCCTT CGCCCTCAGA CGAGTCGGAT CTCCCTTTGG GCCGCCTCCC CGCCTGTCTA 8701 GCTTGACTGA CTGAGATACA GCGTACCTTC AGCTCACAGA CATGATAAGA TACATTGATG 8761 AGTTTGGACA AACCACAACT AGAATGCAGT GAAAAAAATG CTTTATTTGT GAAATTTGTG 8821 ATGCTATTGC TTTATTTGTA ACCATTATAA GCTGCAATAA ACAAGTTAAC AACAACAATT 8881 GCATTCATTT TATGTTTCAG GTTCAGGGGG AGGTGTGGGA GGTTTTTTAA AGCAAGTAAA 8941 ACCTCTACAA ATGTGGTATT GGCCCATCTC TATCGGTATC GTAGCATAAC CCCTTGGGGC 9001 CTCTAAACGG GTCTTGAGGG GTTTTTTGTG CCCCTCGGGC CGGATTGCTA TCTACCGGCA 9061 TTGGCGCAGA AAAAAATGCC TGATGCGACG CTGCGCGTCT TATACTCCCA CATATGCCAG 9121 ATTCAGCAAC GGATACGGCT TCCCCAACTT GCCCACTTCC ATACGTGTCC TCCTTACCAG 9181 AAATTTATCC TTAAGGTCGT CAGCTATCCT GCAGGCGATC TCTCGATTTC GATCAAGACA 9241 TTCCTTTAAT GGTCTTTTCT GGACACCACT AGGGGTCAGA AGTAGTTCAT CAAACTTTCT 9301 TCCCTCCCTA ATCTCATTGG TTACCTTGGG CTATCGAAAC TTAATTAAGC GATCTGCATC 9361 TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC 9421 CCAGTTCCGC CCATTCTCCG CCCCATCGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG 9481 AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG 9541 GCTTTTGCAA AGGAGGTAGC CAACATGATT GAACAAGATG GATTGCACGC AGGTTCTCCC 9601 GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT 9661 GATGCCGCCG TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC 9721 CTGTCCGGTG CCCTGAATGA ACTCCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG 9781 ACGGGCGTTC CTTGCGCAGC TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG 9841 CTATTGGGCG AAGTGCCGGG GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA 9901 GTATCCATCA TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA 9961 TTCGACCACC AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT 10021 GTCGATCAGG ATGATCTGGA CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC 10081 AGGCTCAAGG CGCGGATGCC CGACGGCGAG GATCTCGTCG TGACCCACGG CGATGCCTGC 10141 TTGCCGAATA TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG 10201 GGTGTGGCGG ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT 10261 GGCGGCGAAT GGGCTGACCG CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG 10321 CGCATCGCCT TCTATCGCCT TCTTGACGAG TTCTTCTAGT ATGTAAGCCC TGTGCCTTCT 10381 AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC 10441 ACTCCCACTG TCCTTTCCTA ATAAAATGAG GAAATTGCAT CGCATTGTCT GAGTAGGTGT 10501 CATTCTATTC TGGGGGGTGG GGTGGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT 10561 AGCAGGCATG CTGGGGATGC GGTGGGCTCT ATGGTTAATT AACCAGTCAA GTCAGCTACT 10621 TGGCGAGATC GACTTGTCTG GGTTTCGACT ACGCTCAGAA TTGCGTCAGT CAAGTTCGAT 10681 CTGGTCCTTG CTATTGCACC CGTTCTCCGA TTACGAGTTT CATTTAAATC ATGTGAGCAA 10741 AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC 10801 TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA 10861 CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC 10921 CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT 10981 CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT 11041 GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG 11101 AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA 11161 GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT 11221 ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA 11281 GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT 11341 GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA 11401 CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT 11461 CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA 11521 GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT 11581 CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCATTTAA ATTTCCGAAC TCTCCAAGGC 11641 CCTCGTCGGA AAATCTTCAA ACCTTTCGTC CGATCCATCT TGCAGGCTAC CTCTCGAACG 11701 AACTATCGCA AGTCTCTTGG CCGGCCTTGC GCCTTGGCTA TTGCTTGGCA GCGCCTATCG 11761 CCAGGTATTA CTCCAATCCC GAATATCCGA GATCGGGATC ACCCGAGAGA AGTTCAACCT 11821 ACATCCTCAA TCCCGATCTA TCCGAGATCC GAGGAATATC GAAATCGGGG CGCGCCTGGT 11881 GTACCGAGAA CGATCCTCTC AGTGCGAGTC TCGACGATCC ATATCGTTGC TTGGCAGTCA 11941 GCCAGTCGGA ATCCAGCTTG GGACCCAGGA AGTCCAATCG TCAGATATTG TACTCAAGCC 12001 TGGTCACGGC AGCGTACCGA TCTGTTTAAA CCTAGATATT GATAGTCTGA TCGGTCAACG 12061 TATAATCGAG TCCTAGCTTT TGCAAACATC TATCAAGAGA CAGGATCAGC AGGAGGCTTT 12121 CGCATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC ATTTTGCCTT 12181 CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT 12241 GCGCGAGTGG GTTACATCGA ACTGGATCTC AACAGCGGTA AGATCCTTGA GAGTTTTCGC 12301 CCCGAAGAAC GCTTTCCAAT GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA 12361 TCCCGTATTG ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC 12421 TTGGTTGAGT ATTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC AGTAAGAGAA 12481 TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG CCAACTTACT TCTGACAACG 12541 ATTGGAGGAC CGAAGGAGCT AACCGCTTTT TTGCACAACA TGGGGGATCA TGTAACTCGC 12601 CTTGATCGTT GGGAACCGGA GCTGAATGAA GCCATACCAA ACGACGAGCG TGACACCACG 12661 ATGCCTGTAG CAATGGCAAC AACCTTGCGT AAACTATTAA CTGGCGAACT ACTTACTCTA 12721 GCTTCCCGGC AACAGTTGAT AGACTGGATG GAGGCGGATA AAGTTGCAGG ACCACTTCTG 12781 CGCTCGGCCC TTCCGGCTGG CTGGTTTATT GCTGATAAAT CTGGAGCCGG TGAGCGTGGG 12841 TCTCGCGGTA TCATTGCAGC ACTGGGGCCA GATGGTAAGC CCTCCCGTAT CGTAGTTATC 12901 TACACGACGG GGAGTCAGGC AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT 12961 GCCTCACTGA TTAAGCATTG GTAACCGATT CTAGGTGCAT TGGCGCAGAA AAAAATGCCT 13021 GATGCGACGC TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT 13081 CCCCAACTTG CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGATCGTT 13141 TAAACTCGAC TCTGGCTCTA TCGAATCTCC GTCGTTTCGA GCTTACGCGA ACAGCCGTGG 13201 CGCTCATTTG CTCGTCGGGC ATCGAATCTC GTCAGCTATC GTCAGCTTAC CTTTTTGGCA 13261 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 13321 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 13381 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 13441 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 13501 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 13561 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 13621 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 13681 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 13741 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 13801 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 13861 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 13921 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 13981 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 14041 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 14101 CTCGACACAC CCGCCAGCGGCCGC (SEQ ID NO: 45) LINE-1 plasmid ORF 1-E2A-ORF2 GFP (SEQ ID NO: 46) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGG GAAGCGGACA GTGTACTAAT TATGCTCTCT 1081 TGAAATTGGC TGGAGATGTT GAGAGCAACC CTGGACCTAT GACCGGCTCT AACTCACATA 1141 TCACCATCCT TACACTTAAC ATTAACGGCC TCAACTCAGC TATCAAGCGC CATCGGCTGG 1201 CCAGCTGGAT CAAATCACAG GATCCAAGCG TTTGTTGCAT CCAAGAGACC CACCTGACCT 1261 GTAGAGATAC TCACCGCCTC AAGATCAAGG GATGGCGAAA GATTTATCAG GCGAACGGTA 1321 AGCAGAAGAA AGCCGGAGTC GCAATTCTGG TCTCAGACAA GACGGATTTC AAGCCCACCA 1381 AAATTAAGCG TGATAAGGAA GGTCACTATA TTATGGTGAA AGGCAGCATA CAGCAGGAAG 1441 AACTTACCAT ATTGAACATC TACGCGCCAA ACACCGGCGC ACCTCGCTTT ATCAAACAGG 1501 TCCTGTCCGA TCTGCAGCGA GATCTGGATT CTCATACGTT GATTATGGGT GATTTCAATA 1561 CACCATTGAG CACCCTGGAT CGCAGCACCA GGCAAAAGGT AAATAAAGAC ACGCAAGAGC 1621 TCAATAGCGC ACTGCATCAG GCAGATCTCA TTGATATTTA TCGCACTCTT CATCCTAAGA 1681 GTACCGAGTA CACATTCTTC AGCGCCCCAC ATCATACATA CTCAAAGATC GATCATATCG 1741 TCGGCTCAAA GGCTCTGCTG TCAAAGTGCA AGCGCACAGA GATAATTACA AATTACCTGT 1801 CAGATCATAG CGCGATCAAG CTCGAGCTGA GAATCAAGAA CCTGACCCAG AGCCGGAGTA 1861 CCACTTGGAA GCTTAATAAC CTGCTGCTCA ACGATTATTG GGTCCACAAT GAGATGAAGG 1921 CAGAGATTAA AATGTTCTTC GAAACAAATG AGAATAAGGA TACTACCTAT CAAAACCTTT 1981 GGGATGCCTT TAAGGCCGTC TGCAGAGGCA AGTTCATCGC CCTCAACGCC TATAAAAGAA 2041 AACAAGAGAG ATCTAAGATC GATACTCTCA CCTCTCAGCT GAAGGAGTTG GAGAAACAGG 2101 AACAGACCCA CTCCAAGGCG TCAAGACGGC AGGAGATCAC AAAGATTCGC GCCGAGTTGA 2161 AAGAGATCGA AACCCAAAAG ACTCTTCAGA AAATTAACGA GTCTCGTAGT TGGTTCTTCG 2221 AGCGGATTAA TAAGATAGAC AGACCTCTGG CACGACTGAT TAAGAAGAAG CGCGAAAAGA 2281 ACCAGATTGA TACCATCAAG AACGACAAGG GCGACATCAC TACTGACCCG ACCGAGATCC 2341 AGACCACTAT TCGGGAGTAT TATAAGCATT TGTATGCTAA CAAGCTTGAG AACCTGGAAG 2401 AGATGGACAC TTTTCTGGAT ACCTATACTC TGCCACGGCT TAATCAAGAG GAAGTCGAGT 2461 CCCTCAACCG CCCAATTACA GGAAGCGAGA TTGTGGCCAT AATTAACTCC CTGCCGACAA 2521 AGAAATCTCC TGGTCCGGAC GGGTTTACAG CTGAGTTTTA TCAACGGTAT ATGGAAGAGC 2581 TTGTACCGTT TCTGCTCAAG CTCTTTCAGT CTATAGAAAA GGAAGGCATC TTGCCCAATT 2641 CCTTCTACGA AGCTTCTATA ATACTTATTC CCAAACCAGG ACGCGATACC ACAAAGAAGG 2701 AAAACTTCCG GCCCATTAGT CTCATGAATA TCGACGCTAA AATATTGAAC AAGATTCTCG 2761 CCAACAGAAT CCAACAACAT ATTAAGAAAT TGATACATCA CGACCAGGTG GGGTTTATAC 2821 CTGGCATGCA GGGCTGGTTT AACATCCGGA AGAGTATTAA CGTCATTCAA CACATTAATA 2881 GAGCTAAGGA TAAGAATCAT ATGATCATCT CTATAGACGC GGAAAAGGCA TTCGATAAGA 2941 TTCAGCAGCC ATTTATGCTC AAGACTCTGA ACAAACTCGG CATCGACGGA ACATATTTTA 3001 AGATTATTCG CGCAATTTAC GATAAGCCGA CTGCTAACAT TATCCTTAAC GGCCAAAAGC 3061 TCGAGGCCTT TCCGCTCAAG ACTGGAACCC GCCAAGGCTG TCCCCTCTCC CCGCTTTTGT 3121 TTAATATTGT ACTCGAGGTG CTGGCTAGGG CTATTCGTCA AGAGAAAGAG ATTAAAGGGA 3181 TACAGCTCGG GAAGGAAGAG GTCAAGCTTT CCTTGTTCGC CGATGATATG ATTGTGTACC 3241 TGGAGAATCC TATTGTGTCT GCTCAGAACC TTCTTAAACT TATTTCTAAC TTTAGCAAGG 3301 TCAGCGGCTA TAAGATTAAC GTCCAGAAAT CTCAGGCCTT TCTGTACACA AATAATCGAC 3361 AGACCGAATC CCAGATAATG GGTGAGCTTC CGTTTGTCAT AGCCAGCAAA AGGATAAAGT 3421 ATCTCGGAAT CCAGCTGACA CGAGACGTTA AAGATTTGTT TAAGGAAAAT TACAAGCCTC 3481 TCCTGAAAGA GATTAAGGAA GATACTAATA AGTGGAAGAA TATCCCCTGT TCATGGGTTG 3541 GCAGAATCAA CATAGTGAAG ATGGCAATAC TTCCTAAAGT GATATATCGC TTTAACGCCA 3601 TCCCAATTAA ACTGCCTATG ACCTTCTTTA CGGAGCTCGA GAAAACAACC CTTAAATTTA 3661 TATGGAATCA AAAGAGAGCA AGAATAGCGA AGTCCATCTT GAGCCAGAAG AATAAGGCCG 3721 GTGGGATTAC TTTGCCTGAT TTTAAGTTGT ATTATAAAGC CACAGTAACT AAGACAGCCT 3781 GGTATTGGTA TCAGAATAGA GACATCGACC AGTGGAATCG GACCGAACCA TCAGAGATAA 3841 TGCCCCACAT CTATAATTAC CTTATATTCG ATAAGCCAGA AAAGAATAAA CAGTGGGGCA 3901 AAGACAGCCT CTTCAACAAG TGGTGTTGGG AGAATTGGCT GGCCATATGC CGGAAACTCA 3961 AGCTCGACCC CTTTCTTACA CCCTACACTA AAATCAACAG TAGGTGGATC AAGGACTTGA 4021 ATGTCAAGCC AAAGACTATA AAGACACTGG AAGAGAATCT TGGGATCACA ATACAAGATA 4081 TAGGCGTCGG CAAAGATTTT ATGTCAAAGA CGCCCAAGGC CATGGCCACT AAGGATAAGA 4141 TTGATAAGTG GGACCTTATT AAGCTCAAAA GCTTCTGTAC TGCCAAGGAG ACCACGATCA 4201 GAGTTAATAG GCAGCCCACT ACATGGGAAA AGATTTTCGC CACTTATTCA TCAGATAAGG 4261 GGTTGATAAG CAGAATATAT AACGAGCTGA AGCAGATCTA CAAGAAGAAA ACGAATAATC 4321 CCATCAAGAA GTGGGCAAAA GATATGAACA GGCATTTTAG CAAAGAGGAT ATCTACGCCG 4381 CGAAGAAGCA TATGAAGAAG TGTAGTTCAA GCTTGGCCAT TCGTGAGATG CAGATTAAGA 4441 CGACCATGCG ATACCACCTT ACCCCAGTGA GGATGGCAAT TATCAAGAAA TCTGGCAATA 4501 ATAGATGTTG GCGGGGCTGT GGCGAGATTG GCACCCTGCT CCATTGCTGG TGGGATTGCA 4561 AGCTGGTGCA GCCGCTTTGG AAATCAGTCT GGCGCTTTCT GAGGGACCTC GAGCTTGAGA 4621 TTCCCTTCGA TCCCGCAATT CCCTTGCTCG GAATCTATCC TAACGAATAC AAGAGCTGTT 4681 GTTACAAGGA TACGTGTACC CGGATGTTCA TCGCGGCCTT GTTTACGATA GCTAAGACGT 4741 GGAATCAGCC TAAGTGCCCC ACAATGATCG ATTGGATCAA GAAAATGTGG CATATTTATA 4801 CCATGGAGTA TTACGCAGCA ATTAAGAATG ACGAATTTAT TTCCTTCGTT GGGACCTGGA 4861 TGAAGCTGGA GACTATTATT CTGAGCAAGC TGTCTCAGGA GCAAAAGACA AAGCATAGAA 4921 TCTTCTCTCT CATTGGTGGT AACGACTACA AAGACGATGA CGACAAGTAA AGCGCTTCTA 4981 GAAGTTGTCT CCTCCTGCAC TGACTGACTG ATACAATCGA TTTCTGGATC CGCAGGCCTA 5041 ATCAACCTCT GGATTACAAA ATTTGTGAAA GATTGACTGG TATTCTTAAC TATGTTGCTC 5101 CTTTTACGCT ATGTGGATAC GCTGCTTTAA TGCCTTTGTA TCATGCTATT GCTTCCCGTA 5161 TGGCTTTCAT TTTCTCCTCC TTGTATAAAT CCTGGTTGCT GTCTCTTTAT GAGGAGTTGT 5221 GGCCCGTTGT CAGGCAACGT GGCGTGGTGT GCACTGTGTT TGCTGACGCA ACCCCCACTG 5281 GTTGGGGCAT TGCCACCACC TGTCAGCTCC TTTCCGGGAC TTTCGCTTTC CCCCTCCCTA 5341 TTGCCACGGC GGAACTCATC GCCGCCTGCC TTGCCCGCTG CTGGACAGGG GCTCGGCTGT 5401 TGGGCACTGA CAATTCCGTG GTGTTGTCGG GGAAGCTGAC GTCCTTTCCA TGGCTGCTCG 5461 CCTGTGTTGC CACCTGGATT CTGCGCGGGA CGTCCTTCTG CTACGTCCCT TCGGCCCTCA 5521 ATCCAGCGGA CCTTCCTTCC CGCGAACAAA CGACCCAACA CCCGTGCGTT TTATTCTGTC 5581 TTTTTATTGC CGATCCCCTC AGAAGAACTC GTCAAGAAGG CGATAGAAGG CGATGCGCTG 5641 CGAATCGGGA GCGGCGATAC CGTAAAGCAC GAGGAAGCGG TCAGCCCATT CGCCGCCAAG 5701 CTCTTCAGCA ATATCACGGG TAGCCAACGC TATGTCCTGA TAGCGGTCGG CCGCTTTACT 5761 TGTACAGCTC GTCCATGCCG AGAGTGATCC CGGCGGCGGT CACGAACTCC AGCAGGACCA 5821 TGTGATCGCG CTTCTCGTTG GGGTCTTTGC TCAGGGCGGA CTGGGTGCTC AGGTAGTGGT 5881 TGTCGGGCAG CAGCACGGGG CCGTCGCCGA TGGGGGTGTT CTGCTGGTAG TGGTCGGCCA 5941 GGTGAGTCCA GGAGATGTTT CAGCACTGTT GCCTTTAGTC TCGAGGCAAC TTAGACAACT 6001 GAGTATTGAT CTGAGCACAG CAGGGTGTGA GCTGTTTGAA GATACTGGGG TTGGGGGTGA 6061 AGAAACTGCA GAGGACTAAC TGGGCTGAGA CCCAGTGGCA ATGTTTTAGG GCCTAAGGAA 6121 TGCCTCTGAA AATCTAGATG GACAACTTTG ACTTTGAGAA AAGAGAGGTG GAAATGAGGA 6181 AAATGACTTT TCTTTATTAG ATTTCGGTAG AAAGAACTTT CATCTTTCCC CTATTTTTGT 6241 TATTCGTTTT AAAACATCTA TCTGGAGGCA GGACAAGTAT GGTCATTAAA AAGATGCAGG 6301 CAGAAGGCAT ATATTGGCTC AGTCAAAGTG GGGAACTTTG GTGGCCAAAC ATACATTGCT 6361 AAGGCTATTC CTATATCAGC TGGACACATA TAAAATGCTG CTAATGCTTC ATTACAAACT 6421 TATATCCTTT AATTCCAGAT GGGGGCAAAG TATGTCCAGG GGTGAGGAAC AATTGAAACA 6481 TTTGGGCTGG AGTAGATTTT GAAAGTCAGC TCTGTGTGTG TGTGTGTGTG TGTGTGTGTG 6541 AGAGCGTGTG TTTCTTTTAA CGTTTTCAGC CTACAGCATA CAGGGTTCAT GGTGGCAAGA 6601 AGATAACAAG ATTTAAATTA TGGCCAGTGA CTAGTGCTGC AAGAAGAACA ACTACCTGCA 6661 TTTAATGGGA AAGCAAAATC TCAGGCTTTG AGGGAAGTTA ACATAGGCTT GATTCTGGGT 6721 GGAAGCTGGG TGTGTAGTTA TCTGGAGGCC AGGCTGGAGC TCTCAGCTCA CTATGGGTTC 6781 ATCTTTATTG TCTCCTTTCA TCTCAACAGC TGCACGCTGC CGTCCTCGAT GTTGTGGCGG 6841 ATCTTGAAGT TCACCTTGAT GCCGTTCTTC TGCTTGTCGG CCATGATATA GACGTTGTGG 6901 CTGTTGTAGT TGTACTCCAG CTTGTGCCCC AGGATGTTGC CGTCCTCCTT GAAGTCGATG 6961 CCCTTCAGCT CGATGCGGTT CACCAGGGTG TCGCCCTCGA ACTTCACCTC GGCGCGGGTC 7021 TTGTAGTTGC CGTCGTCCTT GAAGAAGATG GTGCGCTCCT GGACGTAGCC TTCGGGCATG 7081 GCGGACTTGA AGAAGTCGTG CTGCTTCATG TGGTCGGGGT AGCGGCTGAA GCACTGCACG 7141 CCGTAGGTCA GGGTGGTCAC GAGGGTGGGC CAGGGCACGG GCAGCTTGCC GGTGGTGCAG 7201 ATGAACTTCA GGGTCAGCTT GCCGTAGGTG GCATCGCCCT CGCCCTCGCC GGACACGCTG 7261 AACTTGTGGC CGTTTACGTC GCCGTCCAGC TCGACCAGGA TGGGCACCAC CCCGGTGAAC 7321 AGCTCCTCGC CCTTGCTCAC CATGGTGGCG AATTCGAAGC TTGAGCACGA GATCTGAGTC 7381 CGGTAGGCCT AGCGGATCTG ACGGTTCACT AAACCAGCTC TGCTTATATA GACCTCCCAC 7441 CGTACACGCC TACCGCCCAT TTGCGTCAAT GGGGCGGAGT TGTTACGACA TTTTGGAAAG 7501 TCCCGTTGAT TTTGGTGCCA AAACAAACTC CCATTGACGT CAATGGGGTG GAGACTTGGA 7561 AATCCCCGTG AGTCAAACCG CTATCCACGC CCATTGATGT ACTGCCAAAA CCGCATCACC 7621 ATGGTAATAG CGATGACTAA TACGTAGATG TACTGCCAAG TAGGAAAGTC CCATAAGGTC 7681 ATGTACTGGG CATAATGCCA GGCGGGCCAT TTACCGTCAT TGACGTCAAT AGGGGGCGTA 7741 CTTGGCATAT GATACACTTG ATGTACTGCC AAGTGGGCAG TTTACCGTAA ATACTCCACC 7801 CATTGACGTC AATGGAAAGT CCCTATTGGC GTTACTATGG GAACATACGT CATTATTGAC 7861 GTCAATGGGC GGGGGTCGTT GGGCGGTCAG CCAGGCGGGC CATTTACCGT AAGTTATGTA 7921 ACGGGCCTGC TGCCGGCTCT GCGGCCTCTT CCGCGTCTTC GCCTTCGCCC TCAGACGAGT 7981 CGGATCTCCC TTTGGGCCGC CTCCCCGCCT GTCTAGCTTG ACTGACTGAG ATACAGCGTA 8041 CCTTCAGCTC ACAGACATGA TAAGATACAT TGATGAGTTT GGACAAACCA CAACTAGAAT 8101 GCAGTGAAAA AAATGCTTTA TTTGTGAAAT TTGTGATGCT ATTGCTTTAT TTGTAACCAT 8161 TATAAGCTGC AATAAACAAG TTAACAACAA CAATTGCATT CATTTTATGT TTCAGGTTCA 8221 GGGGGAGGTG TGGGAGGTTT TTTAAAGCAA GTAAAACCTC TACAAATGTG GTATTGGCCC 8281 ATCTCTATCG GTATCGTAGC ATAACCCCTT GGGGCCTCTA AACGGGTCTT GAGGGGTTTT 8341 TTGTGCCCCT CGGGCCGGAT TGCTATCTAC CGGCATTGGC GCAGAAAAAA ATGCCTGATG 8401 CGACGCTGCG CGTCTTATAC TCCCACATAT GCCAGATTCA GCAACGGATA CGGCTTCCCC 8461 AACTTGCCCA CTTCCATACG TGTCCTCCTT ACCAGAAATT TATCCTTAAG GTCGTCAGCT 8521 ATCCTGCAGG CGATCTCTCG ATTTCGATCA AGACATTCCT TTAATGGTCT TTTCTGGACA 8581 CCACTAGGGG TCAGAAGTAG TTCATCAAAC TTTCTTCCCT CCCTAATCTC ATTGGTTACC 8641 TTGGGCTATC GAAACTTAAT TAAGCGATCT GCATCTCAAT TAGTCAGCAA CCATAGTCCC 8701 GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TCCGCCCATT CTCCGCCCCA 8761 TCGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT CTGAGCTATT 8821 CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TGCAAAGGAG GTAGCCAACA 8881 TGATTGAACA AGATGGATTG CACGCAGGTT CTCCCGCCGC TTGGGTGGAG AGGCTATTCG 8941 GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC CGCCGTGTTC CGGCTGTCAG 9001 CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC CGGTGCCCTG AATGAACTCC 9061 AGGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG CGTTCCTTGC GCAGCTGTGC 9121 TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT GGGCGAAGTG CCGGGGCAGG 9181 ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC CATCATGGCT GATGCAATGC 9241 GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA CCACCAAGCG AAACATCGCA 9301 TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA TCAGGATGAT CTGGACGAAG 9361 AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT CAAGGCGCGG ATGCCCGACG 9421 GCGAGGATCT CGTCGTGACC CACGGCGATG CCTGCTTGCC GAATATCATG GTGGAAAATG 9481 GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT GGCGGACCGC TATCAGGACA 9541 TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT GACCGCTTCC 9601 TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG 9661 ACGAGTTCTT CTAGTATGTA AGCCCTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC 9721 CCTCCCCCGT GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA 9781 ATGAGGAAAT TGCATCGCAT TGTCTGAGTA GGTGTCATTC TATTCTGGGG GGTGGGGTGG 9841 GGCAGGACAG CAAGGGGGAG GATTGGGAAG ACAATAGCAG GCATGCTGGG GATGCGGTGG 9901 GCTCTATGGT TAATTAACCA GTCAAGTCAG CTACTTGGCG AGATCGACTT GTCTGGGTTT 9961 CGACTACGCT CAGAATTGCG TCAGTCAAGT TCGATCTGGT CCTTGCTATT GCACCCGTTC 10021 TCCGATTACG AGTTTCATTT AAATCATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 10081 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 10141 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 10201 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 10261 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 10321 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 10381 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 10441 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 10501 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 10561 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 10621 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 10681 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 10741 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 10801 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 10861 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 10921 CATAGTTGCA TTTAAATTTC CGAACTCTCC AAGGCCCTCG TCGGAAAATC TTCAAACCTT 10981 TCGTCCGATC CATCTTGCAG GCTACCTCTC GAACGAACTA TCGCAAGTCT CTTGGCCGGC 11041 CTTGCGCCTT GGCTATTGCT TGGCAGCGCC TATCGCCAGG TATTACTCCA ATCCCGAATA 11101 TCCGAGATCG GGATCACCCG AGAGAAGTTC AACCTACATC CTCAATCCCG ATCTATCCGA 11161 GATCCGAGGA ATATCGAAAT CGGGGCGCGC CTGGTGTACC GAGAACGATC CTCTCAGTGC 11221 GAGTCTCGAC GATCCATATC GTTGCTTGGC AGTCAGCCAG TCGGAATCCA GCTTGGGACC 11281 CAGGAAGTCC AATCGTCAGA TATTGTACTC AAGCCTGGTC ACGGCAGCGT ACCGATCTGT 11341 TTAAACCTAG ATATTGATAG TCTGATCGGT CAACGTATAA TCGAGTCCTA GCTTTTGCAA 11401 ACATCTATCA AGAGACAGGA TCAGCAGGAG GCTTTCGCAT GAGTATTCAA CATTTCCGTG 11461 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC 11521 TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCGCG AGTGGGTTAC ATCGAACTGG 11581 ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGCTTT CCAATGATGA 11641 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC 11701 AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTATTCA CCAGTCACAG 11761 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA 11821 GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATTGG AGGACCGAAG GAGCTAACCG 11881 CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA 11941 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACCT 12001 TGCGTAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAG TTGATAGACT 12061 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT 12121 TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG 12181 GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA 12241 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC 12301 CGATTCTAGG TGCATTGGCG CAGAAAAAAA TGCCTGATGC GACGCTGCGC GTCTTATACT 12361 CCCACATATG CCAGATTCAG CAACGGATAC GGCTTCCCCA ACTTGCCCAC TTCCATACGT 12421 GTCCTCCTTA CCAGAAATTT ATCCTTAAGA TCGTTTAAAC TCGACTCTGG CTCTATCGAA 12481 TCTCCGTCGT TTCGAGCTTA CGCGAACAGC CGTGGCGCTC ATTTGCTCGT CGGGCATCGA 12541 ATCTCGTCAG CTATCGTCAG CTTACCTTTT TGGCAGCGAT CGCGGCTCCC GACATCTTGG 12601 ACCATTAGCT CCACAGGTAT CTTCTTCCCT CTAGTGGTCA TAACAGCAGC TTCAGCTACC 12661 TCTCAATTCA AAAAACCCCT CAAGACCCGT TTAGAGGCCC CAAGGGGTTA TGCTATCAAT 12721 CGTTGCGTTA CACACACAAA AAACCAACAC ACATCCATCT TCGATGGATA GCGATTTTAT 12781 TATCTAACTG CTGATCGAGT GTAGCCAGAT CTAGTAATCA ATTACGGGGT CATTAGTTCA 12841 TAGCCCATAT ATGGAGTTCC GCGTTACATA ACTTACGGTA AATGGCCCGC CTGGCTGACC 12901 GCCCAACGAC CCCCGCCCAT TGACGTCAAT AATGACGTAT GTTCCCATAG TAACGCCAAT 12961 AGGGACTTTC CATTGACGTC AATGGGTGGA GTATTTACGG TAAACTGCCC ACTTGGCAGT 13021 ACATCAAGTG TATCATATGC CAAGTACGCC CCCTATTGAC GTCAATGACG GTAAATGGCC 13081 CGCCTGGCAT TATGCCCAGT ACATGACCTT ATGGGACTTT CCTACTTGGC AGTACATCTA 13141 CGTATTAGTC ATCGCTATTA CCATGCTGAT GCGGTTTTGG CAGTACATCA ATGGGCGTGG 13201 ATAGCGGTTT GACTCACGGG GATTTCCAAG TCTCCACCCC ATTGACGTCA ATGGGAGTTT 13261 GTTTTGGCAC CAAAATCAAC GGGACTTTCC AAAATGTCGT AACAACTCCG CCCCATTGAC 13321 GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA AGCAGAGCTG GTTTAGTGAA 13381 CCGTCAGATC AGATCTTTGT CGATCCTACC ATCCACTCGA CACACCCGCC AGCGGCCGC (SEQ ID NO: 46) LINE-1 plasmid ORF 1-P2A-ORF2 GFP (SEQ ID NO: 47) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGG GAAGCGGAGC TACTAACTTC AGCCTGCTGA 1081 AGCAGGCTGG AGACGTGGAG GAGAACCCTG GACCTATGAC CGGCTCTAAC TCACATATCA 1141 CCATCCTTAC ACTTAACATT AACGGCCTCA ACTCAGCTAT CAAGCGCCAT CGGCTGGCCA 1201 GCTGGATCAA ATCACAGGAT CCAAGCGTTT GTTGCATCCA AGAGACCCAC CTGACCTGTA 1261 GAGATACTCA CCGCCTCAAG ATCAAGGGAT GGCGAAAGAT TTATCAGGCG AACGGTAAGC 1321 AGAAGAAAGC CGGAGTCGCA ATTCTGGTCT CAGACAAGAC GGATTTCAAG CCCACCAAAA 1381 TTAAGCGTGA TAAGGAAGGT CACTATATTA TGGTGAAAGG CAGCATACAG CAGGAAGAAC 1441 TTACCATATT GAACATCTAC GCGCCAAACA CCGGCGCACC TCGCTTTATC AAACAGGTCC 1501 TGTCCGATCT GCAGCGAGAT CTGGATTCTC ATACGTTGAT TATGGGTGAT TTCAATACAC 1561 CATTGAGCAC CCTGGATCGC AGCACCAGGC AAAAGGTAAA TAAAGACACG CAAGAGCTCA 1621 ATAGCGCACT GCATCAGGCA GATCTCATTG ATATTTATCG CACTCTTCAT CCTAAGAGTA 1681 CCGAGTACAC ATTCTTCAGC GCCCCACATC ATACATACTC AAAGATCGAT CATATCGTCG 1741 GCTCAAAGGC TCTGCTGTCA AAGTGCAAGC GCACAGAGAT AATTACAAAT TACCTGTCAG 1801 ATCATAGCGC GATCAAGCTC GAGCTGAGAA TCAAGAACCT GACCCAGAGC CGGAGTACCA 1861 CTTGGAAGCT TAATAACCTG CTGCTCAACG ATTATTGGGT CCACAATGAG ATGAAGGCAG 1921 AGATTAAAAT GTTCTTCGAA ACAAATGAGA ATAAGGATAC TACCTATCAA AACCTTTGGG 1981 ATGCCTTTAA GGCCGTCTGC AGAGGCAAGT TCATCGCCCT CAACGCCTAT AAAAGAAAAC 2041 AAGAGAGATC TAAGATCGAT ACTCTCACCT CTCAGCTGAA GGAGTTGGAG AAACAGGAAC 2101 AGACCCACTC CAAGGCGTCA AGACGGCAGG AGATCACAAA GATTCGCGCC GAGTTGAAAG 2161 AGATCGAAAC CCAAAAGACT CTTCAGAAAA TTAACGAGTC TCGTAGTTGG TTCTTCGAGC 2221 GGATTAATAA GATAGACAGA CCTCTGGCAC GACTGATTAA GAAGAAGCGC GAAAAGAACC 2281 AGATTGATAC CATCAAGAAC GACAAGGGCG ACATCACTAC TGACCCGACC GAGATCCAGA 2341 CCACTATTCG GGAGTATTAT AAGCATTTGT ATGCTAACAA GCTTGAGAAC CTGGAAGAGA 2401 TGGACACTTT TCTGGATACC TATACTCTGC CACGGCTTAA TCAAGAGGAA GTCGAGTCCC 2461 TCAACCGCCC AATTACAGGA AGCGAGATTG TGGCCATAAT TAACTCCCTG CCGACAAAGA 2521 AATCTCCTGG TCCGGACGGG TTTACAGCTG AGTTTTATCA ACGGTATATG GAAGAGCTTG 2581 TACCGTTTCT GCTCAAGCTC TTTCAGTCTA TAGAAAAGGA AGGCATCTTG CCCAATTCCT 2641 TCTACGAAGC TTCTATAATA CTTATTCCCA AACCAGGACG CGATACCACA AAGAAGGAAA 2701 ACTTCCGGCC CATTAGTCTC ATGAATATCG ACGCTAAAAT ATTGAACAAG ATTCTCGCCA 2761 ACAGAATCCA ACAACATATT AAGAAATTGA TACATCACGA CCAGGTGGGG TTTATACCTG 2821 GCATGCAGGG CTGGTTTAAC ATCCGGAAGA GTATTAACGT CATTCAACAC ATTAATAGAG 2881 CTAAGGATAA GAATCATATG ATCATCTCTA TAGACGCGGA AAAGGCATTC GATAAGATTC 2941 AGCAGCCATT TATGCTCAAG ACTCTGAACA AACTCGGCAT CGACGGAACA TATTTTAAGA 3001 TTATTCGCGC AATTTACGAT AAGCCGACTG CTAACATTAT CCTTAACGGC CAAAAGCTCG 3061 AGGCCTTTCC GCTCAAGACT GGAACCCGCC AAGGCTGTCC CCTCTCCCCG CTTTTGTTTA 3121 ATATTGTACT CGAGGTGCTG GCTAGGGCTA TTCGTCAAGA GAAAGAGATT AAAGGGATAC 3181 AGCTCGGGAA GGAAGAGGTC AAGCTTTCCT TGTTCGCCGA TGATATGATT GTGTACCTGG 3241 AGAATCCTAT TGTGTCTGCT CAGAACCTTC TTAAACTTAT TTCTAACTTT AGCAAGGTCA 3301 GCGGCTATAA GATTAACGTC CAGAAATCTC AGGCCTTTCT GTACACAAAT AATCGACAGA 3361 CCGAATCCCA GATAATGGGT GAGCTTCCGT TTGTCATAGC CAGCAAAAGG ATAAAGTATC 3421 TCGGAATCCA GCTGACACGA GACGTTAAAG ATTTGTTTAA GGAAAATTAC AAGCCTCTCC 3481 TGAAAGAGAT TAAGGAAGAT ACTAATAAGT GGAAGAATAT CCCCTGTTCA TGGGTTGGCA 3541 GAATCAACAT AGTGAAGATG GCAATACTTC CTAAAGTGAT ATATCGCTTT AACGCCATCC 3601 CAATTAAACT GCCTATGACC TTCTTTACGG AGCTCGAGAA AACAACCCTT AAATTTATAT 3661 GGAATCAAAA GAGAGCAAGA ATAGCGAAGT CCATCTTGAG CCAGAAGAAT AAGGCCGGTG 3721 GGATTACTTT GCCTGATTTT AAGTTGTATT ATAAAGCCAC AGTAACTAAG ACAGCCTGGT 3781 ATTGGTATCA GAATAGAGAC ATCGACCAGT GGAATCGGAC CGAACCATCA GAGATAATGC 3841 CCCACATCTA TAATTACCTT ATATTCGATA AGCCAGAAAA GAATAAACAG TGGGGCAAAG 3901 ACAGCCTCTT CAACAAGTGG TGTTGGGAGA ATTGGCTGGC CATATGCCGG AAACTCAAGC 3961 TCGACCCCTT TCTTACACCC TACACTAAAA TCAACAGTAG GTGGATCAAG GACTTGAATG 4021 TCAAGCCAAA GACTATAAAG ACACTGGAAG AGAATCTTGG GATCACAATA CAAGATATAG 4081 GCGTCGGCAA AGATTTTATG TCAAAGACGC CCAAGGCCAT GGCCACTAAG GATAAGATTG 4141 ATAAGTGGGA CCTTATTAAG CTCAAAAGCT TCTGTACTGC CAAGGAGACC ACGATCAGAG 4201 TTAATAGGCA GCCCACTACA TGGGAAAAGA TTTTCGCCAC TTATTCATCA GATAAGGGGT 4261 TGATAAGCAG AATATATAAC GAGCTGAAGC AGATCTACAA GAAGAAAACG AATAATCCCA 4321 TCAAGAAGTG GGCAAAAGAT ATGAACAGGC ATTTTAGCAA AGAGGATATC TACGCCGCGA 4381 AGAAGCATAT GAAGAAGTGT AGTTCAAGCT TGGCCATTCG TGAGATGCAG ATTAAGACGA 4441 CCATGCGATA CCACCTTACC CCAGTGAGGA TGGCAATTAT CAAGAAATCT GGCAATAATA 4501 GATGTTGGCG GGGCTGTGGC GAGATTGGCA CCCTGCTCCA TTGCTGGTGG GATTGCAAGC 4561 TGGTGCAGCC GCTTTGGAAA TCAGTCTGGC GCTTTCTGAG GGACCTCGAG CTTGAGATTC 4621 CCTTCGATCC CGCAATTCCC TTGCTCGGAA TCTATCCTAA CGAATACAAG AGCTGTTGTT 4681 ACAAGGATAC GTGTACCCGG ATGTTCATCG CGGCCTTGTT TACGATAGCT AAGACGTGGA 4741 ATCAGCCTAA GTGCCCCACA ATGATCGATT GGATCAAGAA AATGTGGCAT ATTTATACCA 4801 TGGAGTATTA CGCAGCAATT AAGAATGACG AATTTATTTC CTTCGTTGGG ACCTGGATGA 4861 AGCTGGAGAC TATTATTCTG AGCAAGCTGT CTCAGGAGCA AAAGACAAAG CATAGAATCT 4921 TCTCTCTCAT TGGTGGTAAC GACTACAAAG ACGATGACGA CAAGTAAAGC GCTTCTAGAA 4981 GTTGTCTCCT CCTGCACTGA CTGACTGATA CAATCGATTT CTGGATCCGC AGGCCTAATC 5041 AACCTCTGGA TTACAAAATT TGTGAAAGAT TGACTGGTAT TCTTAACTAT GTTGCTCCTT 5101 TTACGCTATG TGGATACGCT GCTTTAATGC CTTTGTATCA TGCTATTGCT TCCCGTATGG 5161 CTTTCATTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTC TCTTTATGAG GAGTTGTGGC 5221 CCGTTGTCAG GCAACGTGGC GTGGTGTGCA CTGTGTTTGC TGACGCAACC CCCACTGGTT 5281 GGGGCATTGC CACCACCTGT CAGCTCCTTT CCGGGACTTT CGCTTTCCCC CTCCCTATTG 5341 CCACGGCGGA ACTCATCGCC GCCTGCCTTG CCCGCTGCTG GACAGGGGCT CGGCTGTTGG 5401 GCACTGACAA TTCCGTGGTG TTGTCGGGGA AGCTGACGTC CTTTCCATGG CTGCTCGCCT 5461 GTGTTGCCAC CTGGATTCTG CGCGGGACGT CCTTCTGCTA CGTCCCTTCG GCCCTCAATC 5521 CAGCGGACCT TCCTTCCCGC GAACAAACGA CCCAACACCC GTGCGTTTTA TTCTGTCTTT 5581 TTATTGCCGA TCCCCTCAGA AGAACTCGTC AAGAAGGCGA TAGAAGGCGA TGCGCTGCGA 5641 ATCGGGAGCG GCGATACCGT AAAGCACGAG GAAGCGGTCA GCCCATTCGC CGCCAAGCTC 5701 TTCAGCAATA TCACGGGTAG CCAACGCTAT GTCCTGATAG CGGTCGGCCG CTTTACTTGT 5761 ACAGCTCGTC CATGCCGAGA GTGATCCCGG CGGCGGTCAC GAACTCCAGC AGGACCATGT 5821 GATCGCGCTT CTCGTTGGGG TCTTTGCTCA GGGCGGACTG GGTGCTCAGG TAGTGGTTGT 5881 CGGGCAGCAG CACGGGGCCG TCGCCGATGG GGGTGTTCTG CTGGTAGTGG TCGGCCAGGT 5941 GAGTCCAGGA GATGTTTCAG CACTGTTGCC TTTAGTCTCG AGGCAACTTA GACAACTGAG 6001 TATTGATCTG AGCACAGCAG GGTGTGAGCT GTTTGAAGAT ACTGGGGTTG GGGGTGAAGA 6061 AACTGCAGAG GACTAACTGG GCTGAGACCC AGTGGCAATG TTTTAGGGCC TAAGGAATGC 6121 CTCTGAAAAT CTAGATGGAC AACTTTGACT TTGAGAAAAG AGAGGTGGAA ATGAGGAAAA 6181 TGACTTTTCT TTATTAGATT TCGGTAGAAA GAACTTTCAT CTTTCCCCTA TTTTTGTTAT 6241 TCGTTTTAAA ACATCTATCT GGAGGCAGGA CAAGTATGGT CATTAAAAAG ATGCAGGCAG 6301 AAGGCATATA TTGGCTCAGT CAAAGTGGGG AACTTTGGTG GCCAAACATA CATTGCTAAG 6361 GCTATTCCTA TATCAGCTGG ACACATATAA AATGCTGCTA ATGCTTCATT ACAAACTTAT 6421 ATCCTTTAAT TCCAGATGGG GGCAAAGTAT GTCCAGGGGT GAGGAACAAT TGAAACATTT 6481 GGGCTGGAGT AGATTTTGAA AGTCAGCTCT GTGTGTGTGT GTGTGTGTGT GTGTGTGAGA 6541 GCGTGTGTTT CTTTTAACGT TTTCAGCCTA CAGCATACAG GGTTCATGGT GGCAAGAAGA 6601 TAACAAGATT TAAATTATGG CCAGTGACTA GTGCTGCAAG AAGAACAACT ACCTGCATTT 6661 AATGGGAAAG CAAAATCTCA GGCTTTGAGG GAAGTTAACA TAGGCTTGAT TCTGGGTGGA 6721 AGCTGGGTGT GTAGTTATCT GGAGGCCAGG CTGGAGCTCT CAGCTCACTA TGGGTTCATC 6781 TTTATTGTCT CCTTTCATCT CAACAGCTGC ACGCTGCCGT CCTCGATGTT GTGGCGGATC 6841 TTGAAGTTCA CCTTGATGCC GTTCTTCTGC TTGTCGGCCA TGATATAGAC GTTGTGGCTG 6901 TTGTAGTTGT ACTCCAGCTT GTGCCCCAGG ATGTTGCCGT CCTCCTTGAA GTCGATGCCC 6961 TTCAGCTCGA TGCGGTTCAC CAGGGTGTCG CCCTCGAACT TCACCTCGGC GCGGGTCTTG 7021 TAGTTGCCGT CGTCCTTGAA GAAGATGGTG CGCTCCTGGA CGTAGCCTTC GGGCATGGCG 7081 GACTTGAAGA AGTCGTGCTG CTTCATGTGG TCGGGGTAGC GGCTGAAGCA CTGCACGCCG 7141 TAGGTCAGGG TGGTCACGAG GGTGGGCCAG GGCACGGGCA GCTTGCCGGT GGTGCAGATG 7201 AACTTCAGGG TCAGCTTGCC GTAGGTGGCA TCGCCCTCGC CCTCGCCGGA CACGCTGAAC 7261 TTGTGGCCGT TTACGTCGCC GTCCAGCTCG ACCAGGATGG GCACCACCCC GGTGAACAGC 7321 TCCTCGCCCT TGCTCACCAT GGTGGCGAAT TCGAAGCTTG AGCACGAGAT CTGAGTCCGG 7381 TAGGCCTAGC GGATCTGACG GTTCACTAAA CCAGCTCTGC TTATATAGAC CTCCCACCGT 7441 ACACGCCTAC CGCCCATTTG CGTCAATGGG GCGGAGTTGT TACGACATTT TGGAAAGTCC 7501 CGTTGATTTT GGTGCCAAAA CAAACTCCCA TTGACGTCAA TGGGGTGGAG ACTTGGAAAT 7561 CCCCGTGAGT CAAACCGCTA TCCACGCCCA TTGATGTACT GCCAAAACCG CATCACCATG 7621 GTAATAGCGA TGACTAATAC GTAGATGTAC TGCCAAGTAG GAAAGTCCCA TAAGGTCATG 7681 TACTGGGCAT AATGCCAGGC GGGCCATTTA CCGTCATTGA CGTCAATAGG GGGCGTACTT 7741 GGCATATGAT ACACTTGATG TACTGCCAAG TGGGCAGTTT ACCGTAAATA CTCCACCCAT 7801 TGACGTCAAT GGAAAGTCCC TATTGGCGTT ACTATGGGAA CATACGTCAT TATTGACGTC 7861 AATGGGCGGG GGTCGTTGGG CGGTCAGCCA GGCGGGCCAT TTACCGTAAG TTATGTAACG 7921 GGCCTGCTGC CGGCTCTGCG GCCTCTTCCG CGTCTTCGCC TTCGCCCTCA GACGAGTCGG 7981 ATCTCCCTTT GGGCCGCCTC CCCGCCTGTC TAGCTTGACT GACTGAGATA CAGCGTACCT 8041 TCAGCTCACA GACATGATAA GATACATTGA TGAGTTTGGA CAAACCACAA CTAGAATGCA 8101 GTGAAAAAAA TGCTTTATTT GTGAAATTTG TGATGCTATT GCTTTATTTG TAACCATTAT 8161 AAGCTGCAAT AAACAAGTTA ACAACAACAA TTGCATTCAT TTTATGTTTC AGGTTCAGGG 8221 GGAGGTGTGG GAGGTTTTTT AAAGCAAGTA AAACCTCTAC AAATGTGGTA TTGGCCCATC 8281 TCTATCGGTA TCGTAGCATA ACCCCTTGGG GCCTCTAAAC GGGTCTTGAG GGGTTTTTTG 8341 TGCCCCTCGG GCCGGATTGC TATCTACCGG CATTGGCGCA GAAAAAAATG CCTGATGCGA 8401 CGCTGCGCGT CTTATACTCC CACATATGCC AGATTCAGCA ACGGATACGG CTTCCCCAAC 8461 TTGCCCACTT CCATACGTGT CCTCCTTACC AGAAATTTAT CCTTAAGGTC GTCAGCTATC 8521 CTGCAGGCGA TCTCTCGATT TCGATCAAGA CATTCCTTTA ATGGTCTTTT CTGGACACCA 8581 CTAGGGGTCA GAAGTAGTTC ATCAAACTTT CTTCCCTCCC TAATCTCATT GGTTACCTTG 8641 GGCTATCGAA ACTTAATTAA GCGATCTGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC 8701 CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATCG 8761 CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA 8821 GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAGGAGGTA GCCAACATGA 8881 TTGAACAAGA TGGATTGCAC GCAGGTTCTC CCGCCGCTTG GGTGGAGAGG CTATTCGGCT 8941 ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC 9001 AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTCCAGG 9061 ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG 9121 ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC 9181 TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC 9241 GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG 9301 AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC 9361 ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGCGGATG CCCGACGGCG 9421 AGGATCTCGT CGTGACCCAC GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC 9481 GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG 9541 CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG 9601 TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG 9661 AGTTCTTCTA GTATGTAAGC CCTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTTGCCCCT 9721 CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG 9781 AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC 9841 AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT 9901 CTATGGTTAA TTAACCAGTC AAGTCAGCTA CTTGGCGAGA TCGACTTGTC TGGGTTTCGA 9961 CTACGCTCAG AATTGCGTCA GTCAAGTTCG ATCTGGTCCT TGCTATTGCA CCCGTTCTCC 10021 GATTACGAGT TTCATTTAAA TCATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA 10081 AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA 10141 TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC 10201 CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC 10261 CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG 10321 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA 10381 CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC 10441 GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC 10501 AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGAACAGTAT TTGGTATCTG 10561 CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA 10621 AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA 10681 AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA 10741 CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT 10801 AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG 10861 TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT 10921 AGTTGCATTT AAATTTCCGA ACTCTCCAAG GCCCTCGTCG GAAAATCTTC AAACCTTTCG 10981 TCCGATCCAT CTTGCAGGCT ACCTCTCGAA CGAACTATCG CAAGTCTCTT GGCCGGCCTT 11041 GCGCCTTGGC TATTGCTTGG CAGCGCCTAT CGCCAGGTAT TACTCCAATC CCGAATATCC 11101 GAGATCGGGA TCACCCGAGA GAAGTTCAAC CTACATCCTC AATCCCGATC TATCCGAGAT 11161 CCGAGGAATA TCGAAATCGG GGCGCGCCTG GTGTACCGAG AACGATCCTC TCAGTGCGAG 11221 TCTCGACGAT CCATATCGTT GCTTGGCAGT CAGCCAGTCG GAATCCAGCT TGGGACCCAG 11281 GAAGTCCAAT CGTCAGATAT TGTACTCAAG CCTGGTCACG GCAGCGTACC GATCTGTTTA 11341 AACCTAGATA TTGATAGTCT GATCGGTCAA CGTATAATCG AGTCCTAGCT TTTGCAAACA 11401 TCTATCAAGA GACAGGATCA GCAGGAGGCT TTCGCATGAG TATTCAACAT TTCCGTGTCG 11461 CCCTTATTCC CTTTTTTGCG GCATTTTGCC TTCCTGTTTT TGCTCACCCA GAAACGCTGG 11521 TGAAAGTAAA AGATGCTGAA GATCAGTTGG GTGCGCGAGT GGGTTACATC GAACTGGATC 11581 TCAACAGCGG TAAGATCCTT GAGAGTTTTC GCCCCGAAGA ACGCTTTCCA ATGATGAGCA 11641 CTTTTAAAGT TCTGCTATGT GGCGCGGTAT TATCCCGTAT TGACGCCGGG CAAGAGCAAC 11701 TCGGTCGCCG CATACACTAT TCTCAGAATG ACTTGGTTGA GTATTCACCA GTCACAGAAA 11761 AGCATCTTAC GGATGGCATG ACAGTAAGAG AATTATGCAG TGCTGCCATA ACCATGAGTG 11821 ATAACACTGC GGCCAACTTA CTTCTGACAA CGATTGGAGG ACCGAAGGAG CTAACCGCTT 11881 TTTTGCACAA CATGGGGGAT CATGTAACTC GCCTTGATCG TTGGGAACCG GAGCTGAATG 11941 AAGCCATACC AAACGACGAG CGTGACACCA CGATGCCTGT AGCAATGGCA ACAACCTTGC 12001 GTAAACTATT AACTGGCGAA CTACTTACTC TAGCTTCCCG GCAACAGTTG ATAGACTGGA 12061 TGGAGGCGGA TAAAGTTGCA GGACCACTTC TGCGCTCGGC CCTTCCGGCT GGCTGGTTTA 12121 TTGCTGATAA ATCTGGAGCC GGTGAGCGTG GGTCTCGCGG TATCATTGCA GCACTGGGGC 12181 CAGATGGTAA GCCCTCCCGT ATCGTAGTTA TCTACACGAC GGGGAGTCAG GCAACTATGG 12241 ATGAACGAAA TAGACAGATC GCTGAGATAG GTGCCTCACT GATTAAGCAT TGGTAACCGA 12301 TTCTAGGTGC ATTGGCGCAG AAAAAAATGC CTGATGCGAC GCTGCGCGTC TTATACTCCC 12361 ACATATGCCA GATTCAGCAA CGGATACGGC TTCCCCAACT TGCCCACTTC CATACGTGTC 12421 CTCCTTACCA GAAATTTATC CTTAAGATCG TTTAAACTCG ACTCTGGCTC TATCGAATCT 12481 CCGTCGTTTC GAGCTTACGC GAACAGCCGT GGCGCTCATT TGCTCGTCGG GCATCGAATC 12541 TCGTCAGCTA TCGTCAGCTT ACCTTTTTGG CAGCGATCGC GGCTCCCGAC ATCTTGGACC 12601 ATTAGCTCCA CAGGTATCTT CTTCCCTCTA GTGGTCATAA CAGCAGCTTC AGCTACCTCT 12661 CAATTCAAAA AACCCCTCAA GACCCGTTTA GAGGCCCCAA GGGGTTATGC TATCAATCGT 12721 TGCGTTACAC ACACAAAAAA CCAACACACA TCCATCTTCG ATGGATAGCG ATTTTATTAT 12781 CTAACTGCTG ATCGAGTGTA GCCAGATCTA GTAATCAATT ACGGGGTCAT TAGTTCATAG 12841 CCCATATATG GAGTTCCGCG TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC 12901 CAACGACCCC CGCCCATTGA CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG 12961 GACTTTCCAT TGACGTCAAT GGGTGGAGTA TTTACGGTAA ACTGCCCACT TGGCAGTACA 13021 TCAAGTGTAT CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC 13081 CTGGCATTAT GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT 13141 ATTAGTCATC GCTATTACCA TGCTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA 13201 GCGGTTTGAC TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT 13261 TTGGCACCAA AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA 13321 AATGGGCGGT AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTGGTT TAGTGAACCG 13381 TCAGATCAGA TCTTTGTCGA TCCTACCATC CACTCGACAC ACCCGCCAGC GGCCGC (SEQ ID NO: 47) LINE-1 plasmid ORF1-T2A ORF2 GFP (SEQ ID NO: 48) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGG GAAGCGGAGA GGGCAGAGGA AGTCTGCTAA 1081 CATGCGGTGA CGTCGAGGAG AATCCTGGAC CTATGACCGG CTCTAACTCA CATATCACCA 1141 TCCTTACACT TAACATTAAC GGCCTCAACT CAGCTATCAA GCGCCATCGG CTGGCCAGCT 1201 GGATCAAATC ACAGGATCCA AGCGTTTGTT GCATCCAAGA GACCCACCTG ACCTGTAGAG 1261 ATACTCACCG CCTCAAGATC AAGGGATGGC GAAAGATTTA TCAGGCGAAC GGTAAGCAGA 1321 AGAAAGCCGG AGTCGCAATT CTGGTCTCAG ACAAGACGGA TTTCAAGCCC ACCAAAATTA 1381 AGCGTGATAA GGAAGGTCAC TATATTATGG TGAAAGGCAG CATACAGCAG GAAGAACTTA 1441 CCATATTGAA CATCTACGCG CCAAACACCG GCGCACCTCG CTTTATCAAA CAGGTCCTGT 1501 CCGATCTGCA GCGAGATCTG GATTCTCATA CGTTGATTAT GGGTGATTTC AATACACCAT 1561 TGAGCACCCT GGATCGCAGC ACCAGGCAAA AGGTAAATAA AGACACGCAA GAGCTCAATA 1621 GCGCACTGCA TCAGGCAGAT CTCATTGATA TTTATCGCAC TCTTCATCCT AAGAGTACCG 1681 AGTACACATT CTTCAGCGCC CCACATCATA CATACTCAAA GATCGATCAT ATCGTCGGCT 1741 CAAAGGCTCT GCTGTCAAAG TGCAAGCGCA CAGAGATAAT TACAAATTAC CTGTCAGATC 1801 ATAGCGCGAT CAAGCTCGAG CTGAGAATCA AGAACCTGAC CCAGAGCCGG AGTACCACTT 1861 GGAAGCTTAA TAACCTGCTG CTCAACGATT ATTGGGTCCA CAATGAGATG AAGGCAGAGA 1921 TTAAAATGTT CTTCGAAACA AATGAGAATA AGGATACTAC CTATCAAAAC CTTTGGGATG 1981 CCTTTAAGGC CGTCTGCAGA GGCAAGTTCA TCGCCCTCAA CGCCTATAAA AGAAAACAAG 2041 AGAGATCTAA GATCGATACT CTCACCTCTC AGCTGAAGGA GTTGGAGAAA CAGGAACAGA 2101 CCCACTCCAA GGCGTCAAGA CGGCAGGAGA TCACAAAGAT TCGCGCCGAG TTGAAAGAGA 2161 TCGAAACCCA AAAGACTCTT CAGAAAATTA ACGAGTCTCG TAGTTGGTTC TTCGAGCGGA 2221 TTAATAAGAT AGACAGACCT CTGGCACGAC TGATTAAGAA GAAGCGCGAA AAGAACCAGA 2281 TTGATACCAT CAAGAACGAC AAGGGCGACA TCACTACTGA CCCGACCGAG ATCCAGACCA 2341 CTATTCGGGA GTATTATAAG CATTTGTATG CTAACAAGCT TGAGAACCTG GAAGAGATGG 2401 ACACTTTTCT GGATACCTAT ACTCTGCCAC GGCTTAATCA AGAGGAAGTC GAGTCCCTCA 2461 ACCGCCCAAT TACAGGAAGC GAGATTGTGG CCATAATTAA CTCCCTGCCG ACAAAGAAAT 2521 CTCCTGGTCC GGACGGGTTT ACAGCTGAGT TTTATCAACG GTATATGGAA GAGCTTGTAC 2581 CGTTTCTGCT CAAGCTCTTT CAGTCTATAG AAAAGGAAGG CATCTTGCCC AATTCCTTCT 2641 ACGAAGCTTC TATAATACTT ATTCCCAAAC CAGGACGCGA TACCACAAAG AAGGAAAACT 2701 TCCGGCCCAT TAGTCTCATG AATATCGACG CTAAAATATT GAACAAGATT CTCGCCAACA 2761 GAATCCAACA ACATATTAAG AAATTGATAC ATCACGACCA GGTGGGGTTT ATACCTGGCA 2821 TGCAGGGCTG GTTTAACATC CGGAAGAGTA TTAACGTCAT TCAACACATT AATAGAGCTA 2881 AGGATAAGAA TCATATGATC ATCTCTATAG ACGCGGAAAA GGCATTCGAT AAGATTCAGC 2941 AGCCATTTAT GCTCAAGACT CTGAACAAAC TCGGCATCGA CGGAACATAT TTTAAGATTA 3001 TTCGCGCAAT TTACGATAAG CCGACTGCTA ACATTATCCT TAACGGCCAA AAGCTCGAGG 3061 CCTTTCCGCT CAAGACTGGA ACCCGCCAAG GCTGTCCCCT CTCCCCGCTT TTGTTTAATA 3121 TTGTACTCGA GGTGCTGGCT AGGGCTATTC GTCAAGAGAA AGAGATTAAA GGGATACAGC 3181 TCGGGAAGGA AGAGGTCAAG CTTTCCTTGT TCGCCGATGA TATGATTGTG TACCTGGAGA 3241 ATCCTATTGT GTCTGCTCAG AACCTTCTTA AACTTATTTC TAACTTTAGC AAGGTCAGCG 3301 GCTATAAGAT TAACGTCCAG AAATCTCAGG CCTTTCTGTA CACAAATAAT CGACAGACCG 3361 AATCCCAGAT AATGGGTGAG CTTCCGTTTG TCATAGCCAG CAAAAGGATA AAGTATCTCG 3421 GAATCCAGCT GACACGAGAC GTTAAAGATT TGTTTAAGGA AAATTACAAG CCTCTCCTGA 3481 AAGAGATTAA GGAAGATACT AATAAGTGGA AGAATATCCC CTGTTCATGG GTTGGCAGAA 3541 TCAACATAGT GAAGATGGCA ATACTTCCTA AAGTGATATA TCGCTTTAAC GCCATCCCAA 3601 TTAAACTGCC TATGACCTTC TTTACGGAGC TCGAGAAAAC AACCCTTAAA TTTATATGGA 3661 ATCAAAAGAG AGCAAGAATA GCGAAGTCCA TCTTGAGCCA GAAGAATAAG GCCGGTGGGA 3721 TTACTTTGCC TGATTTTAAG TTGTATTATA AAGCCACAGT AACTAAGACA GCCTGGTATT 3781 GGTATCAGAA TAGAGACATC GACCAGTGGA ATCGGACCGA ACCATCAGAG ATAATGCCCC 3841 ACATCTATAA TTACCTTATA TTCGATAAGC CAGAAAAGAA TAAACAGTGG GGCAAAGACA 3901 GCCTCTTCAA CAAGTGGTGT TGGGAGAATT GGCTGGCCAT ATGCCGGAAA CTCAAGCTCG 3961 ACCCCTTTCT TACACCCTAC ACTAAAATCA ACAGTAGGTG GATCAAGGAC TTGAATGTCA 4021 AGCCAAAGAC TATAAAGACA CTGGAAGAGA ATCTTGGGAT CACAATACAA GATATAGGCG 4081 TCGGCAAAGA TTTTATGTCA AAGACGCCCA AGGCCATGGC CACTAAGGAT AAGATTGATA 4141 AGTGGGACCT TATTAAGCTC AAAAGCTTCT GTACTGCCAA GGAGACCACG ATCAGAGTTA 4201 ATAGGCAGCC CACTACATGG GAAAAGATTT TCGCCACTTA TTCATCAGAT AAGGGGTTGA 4261 TAAGCAGAAT ATATAACGAG CTGAAGCAGA TCTACAAGAA GAAAACGAAT AATCCCATCA 4321 AGAAGTGGGC AAAAGATATG AACAGGCATT TTAGCAAAGA GGATATCTAC GCCGCGAAGA 4381 AGCATATGAA GAAGTGTAGT TCAAGCTTGG CCATTCGTGA GATGCAGATT AAGACGACCA 4441 TGCGATACCA CCTTACCCCA GTGAGGATGG CAATTATCAA GAAATCTGGC AATAATAGAT 4501 GTTGGCGGGG CTGTGGCGAG ATTGGCACCC TGCTCCATTG CTGGTGGGAT TGCAAGCTGG 4561 TGCAGCCGCT TTGGAAATCA GTCTGGCGCT TTCTGAGGGA CCTCGAGCTT GAGATTCCCT 4621 TCGATCCCGC AATTCCCTTG CTCGGAATCT ATCCTAACGA ATACAAGAGC TGTTGTTACA 4681 AGGATACGTG TACCCGGATG TTCATCGCGG CCTTGTTTAC GATAGCTAAG ACGTGGAATC 4741 AGCCTAAGTG CCCCACAATG ATCGATTGGA TCAAGAAAAT GTGGCATATT TATACCATGG 4801 AGTATTACGC AGCAATTAAG AATGACGAAT TTATTTCCTT CGTTGGGACC TGGATGAAGC 4861 TGGAGACTAT TATTCTGAGC AAGCTGTCTC AGGAGCAAAA GACAAAGCAT AGAATCTTCT 4921 CTCTCATTGG TGGTAACGAC TACAAAGACG ATGACGACAA GTAAAGCGCT TCTAGAAGTT 4981 GTCTCCTCCT GCACTGACTG ACTGATACAA TCGATTTCTG GATCCGCAGG CCTAATCAAC 5041 CTCTGGATTA CAAAATTTGT GAAAGATTGA CTGGTATTCT TAACTATGTT GCTCCTTTTA 5101 CGCTATGTGG ATACGCTGCT TTAATGCCTT TGTATCATGC TATTGCTTCC CGTATGGCTT 5161 TCATTTTCTC CTCCTTGTAT AAATCCTGGT TGCTGTCTCT TTATGAGGAG TTGTGGCCCG 5221 TTGTCAGGCA ACGTGGCGTG GTGTGCACTG TGTTTGCTGA CGCAACCCCC ACTGGTTGGG 5281 GCATTGCCAC CACCTGTCAG CTCCTTTCCG GGACTTTCGC TTTCCCCCTC CCTATTGCCA 5341 CGGCGGAACT CATCGCCGCC TGCCTTGCCC GCTGCTGGAC AGGGGCTCGG CTGTTGGGCA 5401 CTGACAATTC CGTGGTGTTG TCGGGGAAGC TGACGTCCTT TCCATGGCTG CTCGCCTGTG 5461 TTGCCACCTG GATTCTGCGC GGGACGTCCT TCTGCTACGT CCCTTCGGCC CTCAATCCAG 5521 CGGACCTTCC TTCCCGCGAA CAAACGACCC AACACCCGTG CGTTTTATTC TGTCTTTTTA 5581 TTGCCGATCC CCTCAGAAGA ACTCGTCAAG AAGGCGATAG AAGGCGATGC GCTGCGAATC 5641 GGGAGCGGCG ATACCGTAAA GCACGAGGAA GCGGTCAGCC CATTCGCCGC CAAGCTCTTC 5701 AGCAATATCA CGGGTAGCCA ACGCTATGTC CTGATAGCGG TCGGCCGCTT TACTTGTACA 5761 GCTCGTCCAT GCCGAGAGTG ATCCCGGCGG CGGTCACGAA CTCCAGCAGG ACCATGTGAT 5821 CGCGCTTCTC GTTGGGGTCT TTGCTCAGGG CGGACTGGGT GCTCAGGTAG TGGTTGTCGG 5881 GCAGCAGCAC GGGGCCGTCG CCGATGGGGG TGTTCTGCTG GTAGTGGTCG GCCAGGTGAG 5941 TCCAGGAGAT GTTTCAGCAC TGTTGCCTTT AGTCTCGAGG CAACTTAGAC AACTGAGTAT 6001 TGATCTGAGC ACAGCAGGGT GTGAGCTGTT TGAAGATACT GGGGTTGGGG GTGAAGAAAC 6061 TGCAGAGGAC TAACTGGGCT GAGACCCAGT GGCAATGTTT TAGGGCCTAA GGAATGCCTC 6121 TGAAAATCTA GATGGACAAC TTTGACTTTG AGAAAAGAGA GGTGGAAATG AGGAAAATGA 6181 CTTTTCTTTA TTAGATTTCG GTAGAAAGAA CTTTCATCTT TCCCCTATTT TTGTTATTCG 6241 TTTTAAAACA TCTATCTGGA GGCAGGACAA GTATGGTCAT TAAAAAGATG CAGGCAGAAG 6301 GCATATATTG GCTCAGTCAA AGTGGGGAAC TTTGGTGGCC AAACATACAT TGCTAAGGCT 6361 ATTCCTATAT CAGCTGGACA CATATAAAAT GCTGCTAATG CTTCATTACA AACTTATATC 6421 CTTTAATTCC AGATGGGGGC AAAGTATGTC CAGGGGTGAG GAACAATTGA AACATTTGGG 6481 CTGGAGTAGA TTTTGAAAGT CAGCTCTGTG TGTGTGTGTG TGTGTGTGTG TGTGAGAGCG 6541 TGTGTTTCTT TTAACGTTTT CAGCCTACAG CATACAGGGT TCATGGTGGC AAGAAGATAA 6601 CAAGATTTAA ATTATGGCCA GTGACTAGTG CTGCAAGAAG AACAACTACC TGCATTTAAT 6661 GGGAAAGCAA AATCTCAGGC TTTGAGGGAA GTTAACATAG GCTTGATTCT GGGTGGAAGC 6721 TGGGTGTGTA GTTATCTGGA GGCCAGGCTG GAGCTCTCAG CTCACTATGG GTTCATCTTT 6781 ATTGTCTCCT TTCATCTCAA CAGCTGCACG CTGCCGTCCT CGATGTTGTG GCGGATCTTG 6841 AAGTTCACCT TGATGCCGTT CTTCTGCTTG TCGGCCATGA TATAGACGTT GTGGCTGTTG 6901 TAGTTGTACT CCAGCTTGTG CCCCAGGATG TTGCCGTCCT CCTTGAAGTC GATGCCCTTC 6961 AGCTCGATGC GGTTCACCAG GGTGTCGCCC TCGAACTTCA CCTCGGCGCG GGTCTTGTAG 7021 TTGCCGTCGT CCTTGAAGAA GATGGTGCGC TCCTGGACGT AGCCTTCGGG CATGGCGGAC 7081 TTGAAGAAGT CGTGCTGCTT CATGTGGTCG GGGTAGCGGC TGAAGCACTG CACGCCGTAG 7141 GTCAGGGTGG TCACGAGGGT GGGCCAGGGC ACGGGCAGCT TGCCGGTGGT GCAGATGAAC 7201 TTCAGGGTCA GCTTGCCGTA GGTGGCATCG CCCTCGCCCT CGCCGGACAC GCTGAACTTG 7261 TGGCCGTTTA CGTCGCCGTC CAGCTCGACC AGGATGGGCA CCACCCCGGT GAACAGCTCC 7321 TCGCCCTTGC TCACCATGGT GGCGAATTCG AAGCTTGAGC ACGAGATCTG AGTCCGGTAG 7381 GCCTAGCGGA TCTGACGGTT CACTAAACCA GCTCTGCTTA TATAGACCTC CCACCGTACA 7441 CGCCTACCGC CCATTTGCGT CAATGGGGCG GAGTTGTTAC GACATTTTGG AAAGTCCCGT 7501 TGATTTTGGT GCCAAAACAA ACTCCCATTG ACGTCAATGG GGTGGAGACT TGGAAATCCC 7561 CGTGAGTCAA ACCGCTATCC ACGCCCATTG ATGTACTGCC AAAACCGCAT CACCATGGTA 7621 ATAGCGATGA CTAATACGTA GATGTACTGC CAAGTAGGAA AGTCCCATAA GGTCATGTAC 7681 TGGGCATAAT GCCAGGCGGG CCATTTACCG TCATTGACGT CAATAGGGGG CGTACTTGGC 7741 ATATGATACA CTTGATGTAC TGCCAAGTGG GCAGTTTACC GTAAATACTC CACCCATTGA 7801 CGTCAATGGA AAGTCCCTAT TGGCGTTACT ATGGGAACAT ACGTCATTAT TGACGTCAAT 7861 GGGCGGGGGT CGTTGGGCGG TCAGCCAGGC GGGCCATTTA CCGTAAGTTA TGTAACGGGC 7921 CTGCTGCCGG CTCTGCGGCC TCTTCCGCGT CTTCGCCTTC GCCCTCAGAC GAGTCGGATC 7981 TCCCTTTGGG CCGCCTCCCC GCCTGTCTAG CTTGACTGAC TGAGATACAG CGTACCTTCA 8041 GCTCACAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG 8101 AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG 8161 CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA 8221 GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATTG GCCCATCTCT 8281 ATCGGTATCG TAGCATAACC CCTTGGGGCC TCTAAACGGG TCTTGAGGGG TTTTTTGTGC 8341 CCCTCGGGCC GGATTGCTAT CTACCGGCAT TGGCGCAGAA AAAAATGCCT GATGCGACGC 8401 TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT CCCCAACTTG 8461 CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGGTCGTC AGCTATCCTG 8521 CAGGCGATCT CTCGATTTCG ATCAAGACAT TCCTTTAATG GTCTTTTCTG GACACCACTA 8581 GGGGTCAGAA GTAGTTCATC AAACTTTCTT CCCTCCCTAA TCTCATTGGT TACCTTGGGC 8641 TATCGAAACT TAATTAAGCG ATCTGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT 8701 AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATCGCTG 8761 ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA 8821 GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA GGAGGTAGCC AACATGATTG 8881 AACAAGATGG ATTGCACGCA GGTTCTCCCG CCGCTTGGGT GGAGAGGCTA TTCGGCTATG 8941 ACTGGGCACA ACAGACAATC GGCTGCTCTG ATGCCGCCGT GTTCCGGCTG TCAGCGCAGG 9001 GGCGCCCGGT TCTTTTTGTC AAGACCGACC TGTCCGGTGC CCTGAATGAA CTCCAGGACG 9061 AGGCAGCGCG GCTATCGTGG CTGGCCACGA CGGGCGTTCC TTGCGCAGCT GTGCTCGACG 9121 TTGTCACTGA AGCGGGAAGG GACTGGCTGC TATTGGGCGA AGTGCCGGGG CAGGATCTCC 9181 TGTCATCTCA CCTTGCTCCT GCCGAGAAAG TATCCATCAT GGCTGATGCA ATGCGGCGGC 9241 TGCATACGCT TGATCCGGCT ACCTGCCCAT TCGACCACCA AGCGAAACAT CGCATCGAGC 9301 GAGCACGTAC TCGGATGGAA GCCGGTCTTG TCGATCAGGA TGATCTGGAC GAAGAGCATC 9361 AGGGGCTCGC GCCAGCCGAA CTGTTCGCCA GGCTCAAGGC GCGGATGCCC GACGGCGAGG 9421 ATCTCGTCGT GACCCACGGC GATGCCTGCT TGCCGAATAT CATGGTGGAA AATGGCCGCT 9481 TTTCTGGATT CATCGACTGT GGCCGGCTGG GTGTGGCGGA CCGCTATCAG GACATAGCGT 9541 TGGCTACCCG TGATATTGCT GAAGAGCTTG GCGGCGAATG GGCTGACCGC TTCCTCGTGC 9601 TTTACGGTAT CGCCGCTCCC GATTCGCAGC GCATCGCCTT CTATCGCCTT CTTGACGAGT 9661 TCTTCTAGTA TGTAAGCCCT GTGCCTTCTA GTTGCCAGCC ATCTGTTGTT TGCCCCTCCC 9721 CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGT CCTTTCCTAA TAAAATGAGG 9781 AAATTGCATC GCATTGTCTG AGTAGGTGTC ATTCTATTCT GGGGGGTGGG GTGGGGCAGG 9841 ACAGCAAGGG GGAGGATTGG GAAGACAATA GCAGGCATGC TGGGGATGCG GTGGGCTCTA 9901 TGGTTAATTA ACCAGTCAAG TCAGCTACTT GGCGAGATCG ACTTGTCTGG GTTTCGACTA 9961 CGCTCAGAAT TGCGTCAGTC AAGTTCGATC TGGTCCTTGC TATTGCACCC GTTCTCCGAT 10021 TACGAGTTTC ATTTAAATCA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA 10081 GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG 10141 ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC 10201 TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC 10261 CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC 10321 GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG 10381 CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC 10441 ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA 10501 GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGA ACAGTATTTG GTATCTGCGC 10561 TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC 10621 CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG 10681 ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC 10741 ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA 10801 TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA 10861 CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT 10921 TGCATTTAAA TTTCCGAACT CTCCAAGGCC CTCGTCGGAA AATCTTCAAA CCTTTCGTCC 10981 GATCCATCTT GCAGGCTACC TCTCGAACGA ACTATCGCAA GTCTCTTGGC CGGCCTTGCG 11041 CCTTGGCTAT TGCTTGGCAG CGCCTATCGC CAGGTATTAC TCCAATCCCG AATATCCGAG 11101 ATCGGGATCA CCCGAGAGAA GTTCAACCTA CATCCTCAAT CCCGATCTAT CCGAGATCCG 11161 AGGAATATCG AAATCGGGGC GCGCCTGGTG TACCGAGAAC GATCCTCTCA GTGCGAGTCT 11221 CGACGATCCA TATCGTTGCT TGGCAGTCAG CCAGTCGGAA TCCAGCTTGG GACCCAGGAA 11281 GTCCAATCGT CAGATATTGT ACTCAAGCCT GGTCACGGCA GCGTACCGAT CTGTTTAAAC 11341 CTAGATATTG ATAGTCTGAT CGGTCAACGT ATAATCGAGT CCTAGCTTTT GCAAACATCT 11401 ATCAAGAGAC AGGATCAGCA GGAGGCTTTC GCATGAGTAT TCAACATTTC CGTGTCGCCC 11461 TTATTCCCTT TTTTGCGGCA TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA 11521 AAGTAAAAGA TGCTGAAGAT CAGTTGGGTG CGCGAGTGGG TTACATCGAA CTGGATCTCA 11581 ACAGCGGTAA GATCCTTGAG AGTTTTCGCC CCGAAGAACG CTTTCCAATG ATGAGCACTT 11641 TTAAAGTTCT GCTATGTGGC GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG 11701 GTCGCCGCAT ACACTATTCT CAGAATGACT TGGTTGAGTA TTCACCAGTC ACAGAAAAGC 11761 ATCTTACGGA TGGCATGACA GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA 11821 ACACTGCGGC CAACTTACTT CTGACAACGA TTGGAGGACC GAAGGAGCTA ACCGCTTTTT 11881 TGCACAACAT GGGGGATCAT GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG 11941 CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACCTTGCGTA 12001 AACTATTAAC TGGCGAACTA CTTACTCTAG CTTCCCGGCA ACAGTTGATA GACTGGATGG 12061 AGGCGGATAA AGTTGCAGGA CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG 12121 CTGATAAATC TGGAGCCGGT GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG 12181 ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG 12241 AACGAAATAG ACAGATCGCT GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACCGATTC 12301 TAGGTGCATT GGCGCAGAAA AAAATGCCTG ATGCGACGCT GCGCGTCTTA TACTCCCACA 12361 TATGCCAGAT TCAGCAACGG ATACGGCTTC CCCAACTTGC CCACTTCCAT ACGTGTCCTC 12421 CTTACCAGAA ATTTATCCTT AAGATCGTTT AAACTCGACT CTGGCTCTAT CGAATCTCCG 12481 TCGTTTCGAG CTTACGCGAA CAGCCGTGGC GCTCATTTGC TCGTCGGGCA TCGAATCTCG 12541 TCAGCTATCG TCAGCTTACC TTTTTGGCAG CGATCGCGGC TCCCGACATC TTGGACCATT 12601 AGCTCCACAG GTATCTTCTT CCCTCTAGTG GTCATAACAG CAGCTTCAGC TACCTCTCAA 12661 TTCAAAAAAC CCCTCAAGAC CCGTTTAGAG GCCCCAAGGG GTTATGCTAT CAATCGTTGC 12721 GTTACACACA CAAAAAACCA ACACACATCC ATCTTCGATG GATAGCGATT TTATTATCTA 12781 ACTGCTGATC GAGTGTAGCC AGATCTAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC 12841 ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA 12901 CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC 12961 TTTCCATTGA CGTCAATGGG TGGAGTATTT ACGGTAAACT GCCCACTTGG CAGTACATCA 13021 AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG 13081 GCATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT 13141 AGTCATCGCT ATTACCATGC TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG 13201 GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG 13261 GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT 13321 GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTGGTTTAG TGAACCGTCA 13381 GATCAGATCT TTGTCGATCC TACCATCCAC TCGACACACC CGCCAGCGGC CGC (SEQ ID NO: 48) LINE-1_ORF2-MCP_MS2_mRNA (SEQ ID NO: 49) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGCCG TCAGACCATC AAGACTAGGA 1081 AGAAACTGCA TCAACTAATG AGCAAAATCA CCAGCTAACA TCATAGTATA CATGACCGGC 1141 TCTAACTCAC ATATCACCAT CCTTACACTT AACATTAACG GCCTCAACTC AGCTATCAAG 1201 CGCCATCGGC TGGCCAGCTG GATCAAATCA CAGGATCCAA GCGTTTGTTG CATCCAAGAG 1261 ACCCACCTGA CCTGTAGAGA TACTCACCGC CTCAAGATCA AGGGATGGCG AAAGATTTAT 1321 CAGGCGAACG GTAAGCAGAA GAAAGCCGGA GTCGCAATTC TGGTCTCAGA CAAGACGGAT 1381 TTCAAGCCCA CCAAAATTAA GCGTGATAAG GAAGGTCACT ATATTATGGT GAAAGGCAGC 1441 ATACAGCAGG AAGAACTTAC CATATTGAAC ATCTACGCGC CAAACACCGG CGCACCTCGC 1501 TTTATCAAAC AGGTCCTGTC CGATCTGCAG CGAGATCTGG ATTCTCATAC GTTGATTATG 1561 GGTGATTTCA ATACACCATT GAGCACCCTG GATCGCAGCA CCAGGCAAAA GGTAAATAAA 1621 GACACGCAAG AGCTCAATAG CGCACTGCAT CAGGCAGATC TCATTGATAT TTATCGCACT 1681 CTTCATCCTA AGAGTACCGA GTACACATTC TTCAGCGCCC CACATCATAC ATACTCAAAG 1741 ATCGATCATA TCGTCGGCTC AAAGGCTCTG CTGTCAAAGT GCAAGCGCAC AGAGATAATT 1801 ACAAATTACC TGTCAGATCA TAGCGCGATC AAGCTCGAGC TGAGAATCAA GAACCTGACC 1861 CAGAGCCGGA GTACCACTTG GAAGCTTAAT AACCTGCTGC TCAACGATTA TTGGGTCCAC 1921 AATGAGATGA AGGCAGAGAT TAAAATGTTC TTCGAAACAA ATGAGAATAA GGATACTACC 1981 TATCAAAACC TTTGGGATGC CTTTAAGGCC GTCTGCAGAG GCAAGTTCAT CGCCCTCAAC 2041 GCCTATAAAA GAAAACAAGA GAGATCTAAG ATCGATACTC TCACCTCTCA GCTGAAGGAG 2101 TTGGAGAAAC AGGAACAGAC CCACTCCAAG GCGTCAAGAC GGCAGGAGAT CACAAAGATT 2161 CGCGCCGAGT TGAAAGAGAT CGAAACCCAA AAGACTCTTC AGAAAATTAA CGAGTCTCGT 2221 AGTTGGTTCT TCGAGCGGAT TAATAAGATA GACAGACCTC TGGCACGACT GATTAAGAAG 2281 AAGCGCGAAA AGAACCAGAT TGATACCATC AAGAACGACA AGGGCGACAT CACTACTGAC 2341 CCGACCGAGA TCCAGACCAC TATTCGGGAG TATTATAAGC ATTTGTATGC TAACAAGCTT 2401 GAGAACCTGG AAGAGATGGA CACTTTTCTG GATACCTATA CTCTGCCACG GCTTAATCAA 2461 GAGGAAGTCG AGTCCCTCAA CCGCCCAATT ACAGGAAGCG AGATTGTGGC CATAATTAAC 2521 TCCCTGCCGA CAAAGAAATC TCCTGGTCCG GACGGGTTTA CAGCTGAGTT TTATCAACGG 2581 TATATGGAAG AGCTTGTACC GTTTCTGCTC AAGCTCTTTC AGTCTATAGA AAAGGAAGGC 2641 ATCTTGCCCA ATTCCTTCTA CGAAGCTTCT ATAATACTTA TTCCCAAACC AGGACGCGAT 2701 ACCACAAAGA AGGAAAACTT CCGGCCCATT AGTCTCATGA ATATCGACGC TAAAATATTG 2761 AACAAGATTC TCGCCAACAG AATCCAACAA CATATTAAGA AATTGATACA TCACGACCAG 2821 GTGGGGTTTA TACCTGGCAT GCAGGGCTGG TTTAACATCC GGAAGAGTAT TAACGTCATT 2881 CAACACATTA ATAGAGCTAA GGATAAGAAT CATATGATCA TCTCTATAGA CGCGGAAAAG 2941 GCATTCGATA AGATTCAGCA GCCATTTATG CTCAAGACTC TGAACAAACT CGGCATCGAC 3001 GGAACATATT TTAAGATTAT TCGCGCAATT TACGATAAGC CGACTGCTAA CATTATCCTT 3061 AACGGCCAAA AGCTCGAGGC CTTTCCGCTC AAGACTGGAA CCCGCCAAGG CTGTCCCCTC 3121 TCCCCGCTTT TGTTTAATAT TGTACTCGAG GTGCTGGCTA GGGCTATTCG TCAAGAGAAA 3181 GAGATTAAAG GGATACAGCT CGGGAAGGAA GAGGTCAAGC TTTCCTTGTT CGCCGATGAT 3241 ATGATTGTGT ACCTGGAGAA TCCTATTGTG TCTGCTCAGA ACCTTCTTAA ACTTATTTCT 3301 AACTTTAGCA AGGTCAGCGG CTATAAGATT AACGTCCAGA AATCTCAGGC CTTTCTGTAC 3361 ACAAATAATC GACAGACCGA ATCCCAGATA ATGGGTGAGC TTCCGTTTGT CATAGCCAGC 3421 AAAAGGATAA AGTATCTCGG AATCCAGCTG ACACGAGACG TTAAAGATTT GTTTAAGGAA 3481 AATTACAAGC CTCTCCTGAA AGAGATTAAG GAAGATACTA ATAAGTGGAA GAATATCCCC 3541 TGTTCATGGG TTGGCAGAAT CAACATAGTG AAGATGGCAA TACTTCCTAA AGTGATATAT 3601 CGCTTTAACG CCATCCCAAT TAAACTGCCT ATGACCTTCT TTACGGAGCT CGAGAAAACA 3661 ACCCTTAAAT TTATATGGAA TCAAAAGAGA GCAAGAATAG CGAAGTCCAT CTTGAGCCAG 3721 AAGAATAAGG CCGGTGGGAT TACTTTGCCT GATTTTAAGT TGTATTATAA AGCCACAGTA 3781 ACTAAGACAG CCTGGTATTG GTATCAGAAT AGAGACATCG ACCAGTGGAA TCGGACCGAA 3841 CCATCAGAGA TAATGCCCCA CATCTATAAT TACCTTATAT TCGATAAGCC AGAAAAGAAT 3901 AAACAGTGGG GCAAAGACAG CCTCTTCAAC AAGTGGTGTT GGGAGAATTG GCTGGCCATA 3961 TGCCGGAAAC TCAAGCTCGA CCCCTTTCTT ACACCCTACA CTAAAATCAA CAGTAGGTGG 4021 ATCAAGGACT TGAATGTCAA GCCAAAGACT ATAAAGACAC TGGAAGAGAA TCTTGGGATC 4081 ACAATACAAG ATATAGGCGT CGGCAAAGAT TTTATGTCAA AGACGCCCAA GGCCATGGCC 4141 ACTAAGGATA AGATTGATAA GTGGGACCTT ATTAAGCTCA AAAGCTTCTG TACTGCCAAG 4201 GAGACCACGA TCAGAGTTAA TAGGCAGCCC ACTACATGGG AAAAGATTTT CGCCACTTAT 4261 TCATCAGATA AGGGGTTGAT AAGCAGAATA TATAACGAGC TGAAGCAGAT CTACAAGAAG 4321 AAAACGAATA ATCCCATCAA GAAGTGGGCA AAAGATATGA ACAGGCATTT TAGCAAAGAG 4381 GATATCTACG CCGCGAAGAA GCATATGAAG AAGTGTAGTT CAAGCTTGGC CATTCGTGAG 4441 ATGCAGATTA AGACGACCAT GCGATACCAC CTTACCCCAG TGAGGATGGC AATTATCAAG 4501 AAATCTGGCA ATAATAGATG TTGGCGGGGC TGTGGCGAGA TTGGCACCCT GCTCCATTGC 4561 TGGTGGGATT GCAAGCTGGT GCAGCCGCTT TGGAAATCAG TCTGGCGCTT TCTGAGGGAC 4621 CTCGAGCTTG AGATTCCCTT CGATCCCGCA ATTCCCTTGC TCGGAATCTA TCCTAACGAA 4681 TACAAGAGCT GTTGTTACAA GGATACGTGT ACCCGGATGT TCATCGCGGC CTTGTTTACG 4741 ATAGCTAAGA CGTGGAATCA GCCTAAGTGC CCCACAATGA TCGATTGGAT CAAGAAAATG 4801 TGGCATATTT ATACCATGGA GTATTACGCA GCAATTAAGA ATGACGAATT TATTTCCTTC 4861 GTTGGGACCT GGATGAAGCT GGAGACTATT ATTCTGAGCA AGCTGTCTCA GGAGCAAAAG 4921 ACAAAGCATA GAATCTTCTC TCTCATTGGT GGTAACGCTT CTAACTTTAC TCAGTTCGTT 4981 CTCGTCGACA ATGGCGGAAC TGGCGACGTG ACTGTCGCCC CAAGCAACTT CGCTAACGGG 5041 ATCGCTGAAT GGATCAGCTC TAACTCGCGT TCACAGGCTT ACAAAGTAAC CTGTAGCGTT 5101 CGTCAGAGCT CTGCGCAGAA TCGCAAATAC ACCATCAAAG TCGAGGTGCC TAAAGGCGCC 5161 TGGCGTTCGT ACTTAAATAT GGAACTAACC ATTCCAATTT TCGCCACGAA TTCCGACTGC 5221 GAGCTTATTG TTAAGGCAAT GCAAGGTCTC CTAAAAGATG GAAACCCGAT TCCCTCAGCA 5281 ATCGCAGCAA ACTCCGGCAT CTACGCCATG GCCAGCAACT TCACCCAGTT CGTGCTGGTG 5341 GACAACGGCG GCACCGGCGA CGTGACCGTG GCCCCCAGCA ACTTCGCCAA CGGCATCGCC 5401 GAGTGGATCA GCAGCAACAG CAGAAGCCAG GCCTACAAGG TGACCTGCAG CGTGAGACAG 5461 AGCAGCGCCC AGAACAGAAA GTACACCATC AAGGTGGAGG TGCCCAAGGG CGCCTGGAGA 5521 AGCTACCTGA ACATGGAGCT GACCATCCCC ATCTTCGCCA CCAACAGCGA CTGCGAGCTG 5581 ATCGTGAAGG CCATGCAGGG CCTGCTGAAG GACGGCAACC CCATCCCCAG CGCCATCGCC 5641 GCCAACAGCG GCATCTACGA CTACAAAGAC GATGACGACA AGTAAAGCAA CCTACAAACG 5701 GGTGGAGGAT CACCCCACCC GACACTTCAC AATCAAGGGG TACAATACAC AAGGGTGGAG 5761 GAACACCCCA CCCTCCAGAC ACATTACACA GAAATCCAAT CAAACAGAAG CACCATCAGG 5821 GCTTCTGCTA CCAAATTTAT CTCAAAAAAC TACAACAAGG AATCACCATC AGGGATTCCC 5881 TGTGCAATAT ACGTCAAACG AGGGCCACGA CGGGAGGACG ATCACGCCTC CCGAATATCG 5941 GCATGTCTGG CTTTCGAATT CAGTGCGTGG AGCATCAGCC CACGCAGCCA ATCAGAGTCG 6001 AATACAAGTC GACTTTCGCG AAGAGCATCA GCCTTCGCGC CATTCTTACA CAAACCACAC 6061 TCTCCCCTAC AGGAACAGCA TCAGCGTTCC TGCCCAGTAC CCAACTCAAG AAAATTTATG 6121 TCCCCATGCA GCATCAGCGC ATGGGCCCCA AGAATACATC CCCAACAAAA TCACATCCGA 6181 GCACCAACAG GGCTCGGAGT GTTGTTTCTT GTCCAACTGG ACAAACCCTC CATGGACCAT 6241 CAGGCCATGG ACTCTCACCA ACAAGACAAA AACTACTCTT CTCGAAGCAG CATCAGCGCT 6301 TCGAAACACT CGAGCATACA TTGTGCCTAT TTCTTGGGTG GACGATCACG CCACCCATGC 6361 TCTCACGAAT TTCAAAACAC GGACAAGGAC GAGCACCACC AGGGCTCGTC GTTCCACGTC 6421 CAATACGATT ACTTACCTTT CGGGATCACG ATCACGGATC CCGCAGCTAC ATCACTTCCA 6481 CTCAGGACAT TCAAGCATGC ACGATCACGG CATGCTCCAC AAGTCTCAAC CACAGAAACT 6541 ACCAAATGGG TTCAGCACCA GCGAACCCAC TCCTACCTCA AACCTCTTCC CACAAAACTG 6601 GCAAGCAGGA TCACCGCTTG CCCATTCCAA CATACCAAAT CAAAAACAAT TACTGGTACA 6661 GCATCAGCGT ACCAGCCCAC ATCTCTCACT ACTATCAAAA ACCAAACCGT TCAGCAACAG 6721 CGAACGGTAC ACACGGAAAA ATCAACTGGT TTACAAATAC GAAAGACGAT CACGCTTTCG 6781 TCCAGCGCAA ACTATTACGA AAAACATCCG ACGGGAAGAG CAACAGCCTT CCCGCGGCGG 6841 AAAACCTCAC AAAAACACGA CAAACGGATG CACGAACACG GCATCCGCCG ACAACCCACA 6901 AACTTACAAC CAGGCAAACG GTGCAGGATC ACCGCACCGT ACATCAAACA CCTCAGATCT 6961 CATGCTTCTA GAAGTTGTCT CCTCCTGCAC TGACTGACTG ATACAATCGA TTTCTGGATC 7021 CGCAGGCCTA ATCAACCTCT GGATTACAAA ATTTGTGAAA GATTGACTGG TATTCTTAAC 7081 TATGTTGCTC CTTTTACGCT ATGTGGATAC GCTGCTTTAA TGCCTTTGTA TCATGCTATT 7141 GCTTCCCGTA TGGCTTTCAT TTTCTCCTCC TTGTATAAAT CCTGGTTGCT GTCTCTTTAT 7201 GAGGAGTTGT GGCCCGTTGT CAGGCAACGT GGCGTGGTGT GCACTGTGTT TGCTGACGCA 7261 ACCCCCACTG GTTGGGGCAT TGCCACCACC TGTCAGCTCC TTTCCGGGAC TTTCGCTTTC 7321 CCCCTCCCTA TTGCCACGGC GGAACTCATC GCCGCCTGCC TTGCCCGCTG CTGGACAGGG 7381 GCTCGGCTGT TGGGCACTGA CAATTCCGTG GTGTTGTCGG GGAAGCTGAC GTCCTTTCCA 7441 TGGCTGCTCG CCTGTGTTGC CACCTGGATT CTGCGCGGGA CGTCCTTCTG CTACGTCCCT 7501 TCGGCCCTCA ATCCAGCGGA CCTTCCTTCC CGCTGAGAGA CACAAAAAAT TCCAACACAC 7561 TATTGCAATG AAAATAAATT TCCTTTATTA GCCAGAAGTC AGATGCTCAA GGGGCTTCAT 7621 GATGTCCCCA TAATTTTTGG CAGAGGGAAA AAGATCTCAG TGGTATTTGT GAGCCAGGGC 7681 ATTGGCCTTC TGATAGGCAG CCTGCACCTG AGGAGTGCGG CCGCTTTACT TGTACAGCTC 7741 GTCCATGCCG AGAGTGATCC CGGCGGCGGT CACGAACTCC AGCAGGACCA TGTGATCGCG 7801 CTTCTCGTTG GGGTCTTTGC TCAGGGCGGA CTGGGTGCTC AGGTAGTGGT TGTCGGGCAG 7861 CAGCACGGGG CCGTCGCCGA TGGGGGTGTT CTGCTGGTAG TGGTCGGCGA GCTGCACGCT 7921 GCCGTCCTCG ATGTTGTGGC GGATCTTGAA GTTCACCTTG ATGCCGTTCT TCTGCTTGTC 7981 GGCCATGATA TAGACGTTGT GGCTGTTGTA GTTGTACTCC AGCTTGTGCC CCAGGATGTT 8041 GCCGTCCTCC TTGAAGTCGA TGCCCTTCAG CTCGATGCGG TTCACCAGGG TGTCGCCCTC 8101 GAACTTCACC TCGGCGCGGG TCTTGTAGTT GCCGTCGTCC TTGAAGAAGA TGGTGCGCTC 8161 CTGGACGTAG CCTTCGGGCA TGGCGGACTT GAAGAAGTCG TGCTGCTTCA TGTGGTCGGG 8221 GTAGCGGCTG AAGCACTGCA CGCCGTAGGT CAGGGTGGTC ACGAGGGTGG GCCAGGGCAC 8281 GGGCAGCTTG CCGGTGGTGC AGATGAACTT CAGGGTCAGC TTGCCGTAGG TGGCATCGCC 8341 CTCGCCCTCG CCGGACACGC TGAACTTGTG GCCGTTTACG TCGCCGTCCA GCTCGACCAG 8401 GATGGGCACC ACCCCGGTGA ACAGCTCCTC GCCCTTGCTC ACCATGGTGG CGGGATCTGA 8461 CGGTTCACTA AACCAGCTCT GCTTATATAG ACCTCCCACC GTACACGCCT ACCGCCCATT 8521 TGCGTCAATG GGGCGGAGTT GTTACGACAT TTTGGAAAGT CCCGTTGATT TTGGTGCCAA 8581 AACAAACTCC CATTGACGTC AATGGGGTGG AGACTTGGAA ATCCCCGTGA GTCAAACCGC 8641 TATCCACGCC CATTGATGTA CTGCCAAAAC CGCATCACCA TGGTAATAGC GATGACTAAT 8701 ACGTAGATGT ACTGCCAAGT AGGAAAGTCC CATAAGGTCA TGTACTGGGC ATAATGCCAG 8761 GCGGGCCATT TACCGTCATT GACGTCAATA GGGGGCGTAC TTGGCATATG ATACACTTGA 8821 TGTACTGCCA AGTGGGCAGT TTACCGTAAA TACTCCACCC ATTGACGTCA ATGGAAAGTC 8881 CCTATTGGCG TTACTATGGG AACATACGTC ATTATTGACG TCAATGGGCG GGGGTCGTTG 8941 GGCGGTCAGC CAGGCGGGCC ATTTACCGTA AGTTATGTAA CGGGCCTGCT GCCGGCTCTG 9001 CGGCCTCTTC CGCGTCTTCG CCTTCGCCCT CAGACGAGTC GGATCTCCCT TTGGGCCGCC 9061 TCCCCGCCTG TCTAGCTTGA CTGACTGAGA TACAGCGTAC CTTCAGCTCA CAGACATGAT 9121 AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA AATGCTTTAT 9181 TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA ATAAACAAGT 9241 T (SEQ ID NO: 49) LINE 1 ORF2-minke mRNA GFP (SEQ ID NO: 50) 1 TAATACGACT CACTATAGGG AGAAGTACTG CCACCATGGG CAAGAAGCAA AATCGCAAGA 61 CGGGGAATTC CAAGACACAA TCCGCTAGCC CACCACCTAA AGAGCGTTCT AGCTCCCCTG 121 CTACTGAGCA GTCCTGGATG GAAAACGACT TCGATGAACT CCGGGAAGAG GGATTTAGGC 181 GATCCAACTA TTCAGAACTC CGCGAAGATA TCCAGACAAA GGGGAAGGAA GTCGAGAATT 241 TCGAGAAGAA CCTCGAGGAG TGCATCACCC GTATCACAAA CACTGAGAAA TGTCTCAAAG 301 AACTCATGGA ACTTAAGACA AAAGCCAGGG AGCTTCGAGA GGAGTGTCGG AGTCTGAGAT 361 CCAGGTGTGA CCAGCTCGAG GAGCGCGTGA GCGCGATGGA AGACGAGATG AACGAGATGA 421 AAAGAGAGGG CAAATTCAGG GAGAAGCGCA TTAAGAGGAA CGAACAGAGT CTGCAGGAGA 481 TTTGGGATTA CGTCAAGAGG CCTAACCTGC GGTTGATCGG CGTCCCCGAG AGCGACGTAG 541 AAAACGGGAC TAAACTGGAG AATACACTTC AAGACATCAT TCAAGAAAAT TTTCCAAACC 601 TGGCTCGGCA AGCTAATGTG CAAATCCAAG AGATCCAACG CACACCCCAG CGGTATAGCT 661 CTCGGCGTGC CACCCCTAGG CATATTATCG TGCGCTTTAC TAAGGTGGAG ATGAAAGAGA 721 AGATGCTGCG AGCCGCTCGG GAAAAGGGAA GGGTGACTTT GAAGGGCAAA CCTATTCGGC 781 TGACGGTTGA CCTTAGCGCC GAGACACTCC AGGCACGCCG GGAATGGGGC CCCATCTTTA 841 ATATCCTGAA GGAGAAGAAC TTCCAGCCAC GAATCTCTTA CCCTGCAAAG TTGAGTTTTA 901 TCTCCGAGGG TGAGATTAAG TATTTCATCG ATAAACAGAT GCTGCGAGAC TTCGTGACAA 961 CTCGCCCAGC TCTCAAGGAA CTGCTCAAAG AGGCTCTTAA TATGGAGCGC AATAATAGAT 1021 ATCAACCCTT GCAGAACCAC GCAAAGATGT GAGACAGCCG TCAGACCATC AAGACTAGGA 1081 AGAAACTGCA TCAACTAATG AGCAAAATCA CCAGCTAACA TCATAGTATA CATGGTCATA 1141 GGAACTTACA TTTCGATTAT TACCTTAAAC GTGAATGGGT TAAATGCCCC AACCAAGAGA 1201 CATCGGCTGG CTGAATGGAT TCAGAAACAG GACCCCTATA TTTGCTGTCT GCAGGAGACC 1261 CACTTCCGTC CTCGCGACAC ATACAGACTG AAAGTGAGGG GCTGGAAAAA GATCTTCCAT 1321 GCCAATGGAA ATCAAAAGAA AGCTGGAGTG GCTATTCTCA TCTCAGATAA AATTGACTTC 1381 AAAATAAAGA ATGTTACTCG AGATAAGGAG GGACACTACA TAATGATCCA GGGGTCCATC 1441 CAAGAAGAGG ATATAACTAT TATTAATATT TATGCACCCA ACATTGGCGC CCCTCAGTAC 1501 ATCAGGCAGC TGCTTACAGC TATCAAGGAG GAAATCGACA GTAACACGAT TATCGTGGGG 1561 GACTTTAACA CCAGCCTTAC TCCGATGGAT AGATCATCCA AAATGAAAAT AAATAAGGAA 1621 ACAGAGGCTC TTAATGACAC CATTGACCAG ATAGATCTGA TTGATATATA TAGGACATTC 1681 CATCCAAAAA CTGCCGATTA CACTTTCTTC AGCAGTGCGC ATGGAACCTT CTCCAGGATA 1741 GATCACATCT TGGGTCACAA AAGTAGCCTC AGTAAGTTTA AGAAAATTGA AATCATTAGC 1801 AGCATCTTTT CTGACCATAA CGCTATGCGC CTGGAGATGA ATCACAGGGA GAAGAACGTA 1861 AAGAAGACAA ACACCTGGAG GCTGAACAAT ACGCTGCTAA ATAACCAAGA GATCACTGAG 1921 GAAATCAAAC AGGAAATAAA AAAATACTTG GAGACAAATG ACAATGAAAA CACGACCACC 1981 CAGAACTTGT GGGATGCAGC TAAAGCGGTT CTGAGAGGGA AGTTTATAGC TATTCAAGCC 2041 TACCTTAAGA AACAGGAAAA ATCTCAAGTG AACAATTTGA CCTTACACCT AAAGAAACTG 2101 GAGAAGGAGG AGCAGACCAA ACCCAAAGTG AGCAGGAGGA AAGAAATCAT CAAGATCAGA 2161 GCCGAAATCA ATGAAATAGA AACTAAGAAG ACAATTGCCA AGATCAATAA AACTAAATCC 2221 TGGTTCTTTG AGAAGATCAA CAAAATTGAT AAGCCATTAG CCAGACTCAT CAAGAAAAAG 2281 AGGGAGAGGA CTCAGATCAA TAAGATCAGA AATGAGAAAG GGGAAGTTAC AACCGACACC 2341 GCGGAGATTC AGAACATCCT GAGAGACTAC TACAAGCAAC TTTATGCCAA TAAAATGGAC 2401 AACCTGGAAG AAATGGACAA ATTCCTGGAA AGGTATAACC TTCCCCGGCT GAACCAGGAG 2461 GAGACTGAAA ATATCAACCG CCCAATCACA AGTAATGAGA TTGAGACTGT GATTAAGAAT 2521 CTTCCAACTA ACAAAAGTCC CGGCCCCGAT GGCTTCACAG GTGAATTCTA TCAGACCTTT 2581 CGGGAGGAGT TGACACCCAT CCTTCTCAAG CTCTTCCAAA AAATTGCAGA GGAGGGCACA 2641 CTCCCGAACT CATTCTATGA GGCCACCATC ACCCTGATCC CAAAGCCCGA CAAGGACACT 2701 ACAAAGAAAG AAAATTACCG ACCAATTTCC CTGATGAATA TCGATGCCAA GATCCTCAAC 2761 AAAATCTTGG CAAACAGAAT CCAGCAGCAC ATTAAGAGGA TCATACACCA CGATCAGGTG 2821 GGCTTTATCC CGGGGATGCA AGGATTCTTC AATATCCGCA AATCAATCAA TGTGATCCAC 2881 CATATTAACA AGTTGAAGAA GAAGAACCAT ATGATCATCT CCATCGATGC AGAGAAAGCT 2941 TTTGACAAAA TTCAACACCC ATTTATGATC AAAACTCTCC AGAAGGTGGG CATCGAGGGG 3001 ACCTACCTCA ACATAATTAA GGCCATCTAT GATAAGCCCA CAGCCAACAT CATTCTCAAT 3061 GGTGAAAAGC TGAAGGCATT TCCTCTGCGG TCCGGAACGA GACAGGGATG TCCTCTCTCT 3121 CCTCTTCTGT TCAACATCGT TCTGGAAGTC CTAGCCACCG CTATCCGCGA GGAAAAGGAA 3181 ATTAAAGGCA TACAGATTGG AAAGGAAGAG GTAAAACTGT CTCTGTTTGC GGATGATATG 3241 ATACTGTACA TAGAGAATCC TAAAACTGCC ACCCGGAAGC TGTTGGAGCT AATTAATGAG 3301 TATGGTAAGG TCGCCGGTTA CAAGATTAAT GCTCAGAAGT CTCTTGCTTT CCTGTACACT 3361 AATGATGAAA AGTCTGAACG GGAAATTATG GAGACACTCC CCTTTACCAT TGCAACCAAA 3421 CGTATTAAAT ACCTTGGCAT TAACCTGCCT AAGGAGACAA AAGACCTGTA TGCTGAAAAC 3481 TATAAGACAC TGATGAAAGA GATTAAAGAT GATACCAACC GGTGGCGGGA TATCCCATGT 3541 TCTTGGATTG GCAGAATCAA CATTGTGAAG ATGAGCATCC TGCCCAAGGC CATCTACAGA 3601 TTCAATGCCA TCCCTATCAA ATTACCTATG GCATTTTTTA CGGAGCTGGA ACAGATCATC 3661 TTAAAATTTG TGTGGCGCCA CAAGCGGCCC CGAATCGCCA AAGCGGTCTT GAGGCAGAAG 3721 AATGGCGCTG GGGGAATCCG ACTCCCTGAC TTCAGATTGT ACTACAAAGC TACCGTCATC 3781 AAGACAATCT GGTACTGGCA CAAGAACAGA AACATCGATC AGTGGAACAA GATCGAAAGC 3841 CCTGAGATTA ACCCCCGCAC CTATGGTCAA CTGATCTATG ACAAAGGGGG CAAGGATATA 3901 CAATGGCGCA AGGACAGCCT CTTCAATAAG TGGTGCTGGG AAAACTGGAC AGCCACCTGC 3961 AAGCGTATGA AGCTGGAGTA CTCCCTGACA CCATACACAA AAATAAACTC AAAGTGGATT 4021 CGAGACCTCA ATATTCGGCT GGACACTATA AAACTCCTGG AGGAGAACAT TGGGCGTACA 4081 CTCTTTGACA TTAATCATAG CAAGATCTTT TTCGATCCCC CTCCTCGTGT AATGGAAATA 4141 AAAACAAAAA TAAACAAGTG GGATCTGATG AAACTTCAGA GCTTTTGCAC CGCAAAGGAG 4201 ACCATAAACA AGACGAAGCG CCAACCCTCA GAATGGGAGA AAATATTTGC GAATGAGTCT 4261 ACGGACAAAG GCTTAATCTC CAAAATATAT AAGCAGCTCA TTCAGCTCAA TATCAAGGAA 4321 ACAAACACCC CGATCCAAAA GTGGGCAGAG GACCTAAATC GGCATTTCTC CAAGGAAGAC 4381 ATCCAGACGG CCACGAAGCA CATGAAGCGA TGCTCAACTT CCCTGATTAT TCGCGAAATG 4441 CAGATCAAGA CTACTATGCG CTATCACCTC ACTCCTGTTC GGATGGGCAT CATCCGGAAA 4501 TCTACAAACA ACAAGTGCTG GAGAGGGTGT GGCGAAAAGG GAACCCTCTT GCATTGTTGG 4561 TGGGAGTGTA AGTTGATCCA GCCACTATGG CGGACCATAT GGAGGTTCCT TAAAAAACTG 4621 AAGATTGAGC TGCCATATGA CCCAGCAATC CCACTGCTGG GCATATACCC GGAGAAAACC 4681 GTGATTCAGA AAGACACTTG CACCCGAATG TTCATTGCAG CATTGTTTAC AATAGCCAGG 4741 TCATGGAAGC AGCCTAAGTG CCCCTCGACA GACGAGTGGA TCAAGAAGAT GTGGTACATT 4801 TATACTATGG AATATTACAG CGCCATCAAA CGCAACGAAA TTGGGTCTTT TCTGGAGACG 4861 TGGATGGATC TAGAGACTGT CATCCAGAGT GAGGTAAGTC AGAAAGAGAA GAACAAATAT 4921 CGTATTTTAA CGCATATTTG TGGAACCTGG AAGAATGGTA CAGATGAGCC GGTCTGCCGA 4981 ACCGAGATTG AGACCCAGAT GGACTACAAA GACGATGACG ACAAGTGAAG CGCTTCTAGA 5041 AGTTGTCTCC TCCTGCACTG ACTGACTGAT ACAATCGATT TCTGGATCCG CAGGCCTAAT 5101 CAACCTCTGG ATTACAAAAT TTGTGAAAGA TTGACTGGTA TTCTTAACTA TGTTGCTCCT 5161 TTTACGCTAT GTGGATACGC TGCTTTAATG CCTTTGTATC ATGCTATTGC TTCCCGTATG 5221 GCTTTCATTT TCTCCTCCTT GTATAAATCC TGGTTGCTGT CTCTTTATGA GGAGTTGTGG 5281 CCCGTTGTCA GGCAACGTGG CGTGGTGTGC ACTGTGTTTG CTGACGCAAC CCCCACTGGT 5341 TGGGGCATTG CCACCACCTG TCAGCTCCTT TCCGGGACTT TCGCTTTCCC CCTCCCTATT 5401 GCCACGGCGG AACTCATCGC CGCCTGCCTT GCCCGCTGCT GGACAGGGGC TCGGCTGTTG 5461 GGCACTGACA ATTCCGTGGT GTTGTCGGGG AAGCTGACGT CCTTTCCATG GCTGCTCGCC 5521 TGTGTTGCCA CCTGGATTCT GCGCGGGACG TCCTTCTGCT ACGTCCCTTC GGCCCTCAAT 5581 CCAGCGGACC TTCCTTCCCG CTGAGAGACA CAAAAAATTC CAACACACTA TTGCAATGAA 5641 AATAAATTTC CTTTATTAGC CAGAAGTCAG ATGCTCAAGG GGCTTCATGA TGTCCCCATA 5701 ATTTTTGGCA GAGGGAAAAA GATCTCAGTG GTATTTGTGA GCCAGGGCAT TGGCCTTCTG 5761 ATAGGCAGCC TGCACCTGAG GAGTGCGGCC GCTTTACTTG TACAGCTCGT CCATGCCGAG 5821 AGTGATCCCG GCGGCGGTCA CGAACTCCAG CAGGACCATG TGATCGCGCT TCTCGTTGGG 5881 GTCTTTGCTC AGGGCGGACT GGGTGCTCAG GTAGTGGTTG TCGGGCAGCA GCACGGGGCC 5941 GTCGCCGATG GGGGTGTTCT GCTGGTAGTG GTCGGCGAGC TGCACGCTGC CGTCCTCGAT 6001 GTTGTGGCGG ATCTTGAAGT TCACCTTGAT GCCGTTCTTC TGCTTGTCGG CCATGATATA 6061 GACGTTGTGG CTGTTGTAGT TGTACTCCAG CTTGTGCCCC AGGATGTTGC CGTCCTCCTT 6121 GAAGTCGATG CCCTTCAGCT CGATGCGGTT CACCAGGGTG TCGCCCTCGA ACTTCACCTC 6181 GGCGCGGGTC TTGTAGTTGC CGTCGTCCTT GAAGAAGATG GTGCGCTCCT GGACGTAGCC 6241 TTCGGGCATG GCGGACTTGA AGAAGTCGTG CTGCTTCATG TGGTCGGGGT AGCGGCTGAA 6301 GCACTGCACG CCGTAGGTCA GGGTGGTCAC GAGGGTGGGC CAGGGCACGG GCAGCTTGCC 6361 GGTGGTGCAG ATGAACTTCA GGGTCAGCTT GCCGTAGGTG GCATCGCCCT CGCCCTCGCC 6421 GGACACGCTG AACTTGTGGC CGTTTACGTC GCCGTCCAGC TCGACCAGGA TGGGCACCAC 6481 CCCGGTGAAC AGCTCCTCGC CCTTGCTCAC CATGGTGGCG GGATCTGACG GTTCACTAAA 6541 CCAGCTCTGC TTATATAGAC CTCCCACCGT ACACGCCTAC CGCCCATTTG CGTCAATGGG 6601 GCGGAGTTGT TACGACATTT TGGAAAGTCC CGTTGATTTT GGTGCCAAAA CAAACTCCCA 6661 TTGACGTCAA TGGGGTGGAG ACTTGGAAAT CCCCGTGAGT CAAACCGCTA TCCACGCCCA 6721 TTGATGTACT GCCAAAACCG CATCACCATG GTAATAGCGA TGACTAATAC GTAGATGTAC 6781 TGCCAAGTAG GAAAGTCCCA TAAGGTCATG TACTGGGCAT AATGCCAGGC GGGCCATTTA 6841 CCGTCATTGA CGTCAATAGG GGGCGTACTT GGCATATGAT ACACTTGATG TACTGCCAAG 6901 TGGGCAGTTT ACCGTAAATA CTCCACCCAT TGACGTCAAT GGAAAGTCCC TATTGGCGTT 6961 ACTATGGGAA CATACGTCAT TATTGACGTC AATGGGCGGG GGTCGTTGGG CGGTCAGCCA 7021 GGCGGGCCAT TTACCGTAAG TTATGTAACG GGCCTGCTGC CGGCTCTGCG GCCTCTTCCG 7081 CGTCTTCGCC TTCGCCCTCA GACGAGTCGG ATCTCCCTTT GGGCCGCCTC CCCGCCTGTC 7141 TAGCTTGACT GACTGAGATA CAGCGTACCT TCAGCTCACA GACATGATAA GATACATTGA 7201 TGAGTTTGGA CAAACCACAA CTAGAATGCA GTGAAAAAAA TGCTTTATTT GTGAAATTTG 7261 TGATGCTATT GCTTTATTTG TAACCATTAT AAGCTGCAAT AAACAAGTT (SEQ ID NO: 50)

Example 17. Enriching Stably Retrotransposed Cells

In an effort to increase the cell yield having stably integrated nucleic acid sequence a method of sorting and culturing was attempted, as described in this example. 293T cells were electroporated with LINE1-GFP mRNA produced by IVT and cultured in vitro for at least 3 days. Expression of GFP was determined periodically using flow cytometry, as shown in FIG. 40. Genomic integration per genome was evaluated using quantitative PCR. Interpolations of nucleic acid encoding GFP in the genome per genome were evaluated using standard curves for GFP and a housekeeping gene (FAU). In a sorting and enrichment culture of GFP positive cells, shown in FIG. 40, it was evident that integration was stable for multiple cell passages (at least 18 days post EP), and considerable enrichment was possible. GFP expression was detectable in ˜1% of 293T cells 5 days post-EP. GFP+ cells were enriched to ˜28% after first sorting and was further enriched up to ˜74% of cells after 2nd sorting. (FIG. 40, FIG. 41C).

Standard curves and exemplary quantitation of genomic integrations are shown in FIGS. 41A and 41B respectively. FIG. 41C shows average number of GFP integrations per genome when gated at 10{circumflex over ( )}3 units of GFP fluorescence intensity and at 10{circumflex over ( )}4 units of GFP fluorescence intensity.

Example 18. Titration of mRNA Concentration for Increased Transposon Mediated Integration

The concentration of LINE1-GFP mRNA used for electroporation was titrated for optimum genomic integration per cell in different cell types, 293T cells, K562 and THP-1 cells (FIGS. 42-46). 100, 500, 1000, 1500 and 2000 ng/μL of mRNA were tested for GFP expression and number of integrations per cell. Concentrations higher than 1000 ng/μl cause cell death. From the results shown in FIGS. 42, 43 and 44 that 1000 ng/μl causes a higher and long-term expression of GFP encoded by the retrotransposed integrated nucleic acid. Integrated DNA encoded protein expression starts to be detectable at day 3 and peaks around day 6-7 (FIG. 45). However, genomic integration and expression of the LINE-1 GFP mRNA in K562 and THP-1 was quite low; integration was detected at about 0.067-0.155 per cell in K562 cells (FIG. 46). (THP-1 data not shown). Higher LINE1-GFP mRNA concentrations (1500 and 2000 ng/μl) caused cell death in these cells. GFP mRNA expression in PD-0015 monocytes was detected at day 3 post electroporation, with detectable integration per cell. (FIG. 47). Steps were to be taken for more extensive DNase 1 treatment, and test mRNA batches were to be evaluated for residual plasmid before electroporation. Accuracy in determination of integration levels in the genome could be improved by first enriching for integrated DNA sequence by PCR followed by paired end sequencing leading to mapping the integration sites within the genome. Next generation sequencing is considered the gold standard in this respect, which involves gDNA extraction→shearing by sonication→DNA linkers ligated onto DNA ends→nested PCR (1: one primer for linker, second to integrated DNA, 2: Illumina sequencing adapters added)→paired-end sequencing.

Example 19. Improvement of Integration Efficiency by Knockdown of Candidates that Prevent Transposon Mediated Integration

In this example, a number of endogenous candidates were knocked down using siRNA to determine if the knockdown could result in higher integration of test nucleic acid encoding GFP. Candidates included inhibitors of LINE1 retrotransposition: ADAR1, ADAR2 (ADAR1B), APOBEC3C, BRCA1, let-7 miRNA, RNase L, TASOR (HUSH complex). siRNAs (3 per target candidate) were made, electroporated in test cells along with LINE1-GFP mRNA and tested for alteration of the LINE-1 GFP expression by flow cytometry and its genome integration by qPCR and a cocktail of the siRNA that help increase LINE-1 GFP integration and expression was selected for further titration. Results from the different siRNAs tested are shown in FIGS. 48-51. Knockdown of ADAR1, BRCA and RNASEL tested individually induced about 2-fold increase in integration of LINE1-GFP. ADAR2 and APOEBEC3C each led to less than 1.5-fold increase, and let7 miRNA and TASOR each led to no increase. In the study shown in FIG. 48, LINE-1 GFP (2000 ng/μL) was electroporated with an siBRCA at 100, 200 and 300 ng/μL in 293 cells, data shown at 4 days post electroporation. With 100 ng/μL, the integration rate was approximately ˜0.06 GFP copies per cell, and siBRCA1_s459 (100 ng/μl) increases integration by ˜2-fold. Data shown in FIG. 49 demonstrates that at day 6 post electroporation, each of siRNASEL and siADAR1 siRNAs separately increased integration about 2-fold. On the other hand, siAPOBEC3C_s2617 increases GFP integration <1.5-fold (FIG. 50) at 6 days post electroporation.

TABLE 11 Effect of specific knockdowns on genomic integration rate. Target GFP integration fold change in 293T cells ADAR1 ~2 fold increase ADAR2 <1.5-fold increase APOEBEC3C <1.5-fold increase BRCA ~2 fold increase Let7 miRNA No increase RNASEL ~2 fold increase TASOR (Hush complex) No increase

siRNA against ADAR, APOEBEC3C, BRCA and RNASEL were chosen for the siRNA cocktail. Using 1000 ng/μL and 1500 ng/μL LINE1-GFP mRNA in two sets of experiments, the concentration of the siRNAs for electroporation was titrated next. It was observed that LINE1-GFP mRNA at 1500 ng/μL was slightly toxic (FIG. 51). With 1000 ng/μL, 75 ng/μL of each siRNA resulted in ˜5-fold improvement of integration of GFP in 293T cells. These results were highly encouraging and support further development. Results from a similar experiment in K562 cells are shown in FIG. 52. 

1-30. (canceled)
 31. A method of expressing an exogenous human therapeutic polypeptide from a genomically integrated DNA sequence of a target human cell, the method comprising: (a) contacting an isolated RNA molecule to the target human cell, wherein the target human cell uptakes the isolated RNA molecule and wherein the isolated RNA molecule comprises: (i) an RNA sequence that is a reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide, and (ii) a human mobile genetic element comprising an RNA sequence encoding a polypeptide with target-primed reverse transcription (TPRT) activity; (b) translating the human mobile genetic element to produce the polypeptide with TPRT activity; (c) reverse transcribing the RNA sequence that is the reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide via the TPRT activity of the polypeptide translated in step (b), thereby producing the DNA sequence encoding the exogenous human therapeutic polypeptide; (d) integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA of the target human cell at a target site; and (e) after step (d), expressing the exogenous human therapeutic polypeptide from the genomically integrated DNA sequence encoding the exogenous human therapeutic polypeptide in the target human cell.
 32. The method of claim 31, wherein the isolated RNA molecule is a synthetic or in vitro transcribed RNA molecule.
 33. The method of claim 31, wherein the isolated RNA molecule is purified.
 34. The method of claim 31, wherein the exogenous human therapeutic polypeptide is expressed in at least 2% of the cells in the target human cell population.
 35. The method of claim 31, wherein the target human cell is an immune cell selected from the group consisting of a T cell, a B cell, a myeloid cell, a monocyte, a macrophage and a dendritic cell.
 36. The method of claim 31, wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises a human ORF2p or a functional fragment thereof.
 37. The method of claim 31, wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises a nuclear localization signal (NLS).
 38. The method of claim 36, wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises human ORF1p or a functional fragment thereof.
 39. The method of claim 38, wherein the human ORF1p or the functional fragment thereof and the human ORF2p or the functional fragment thereof are translated from different open reading frames of the same isolated RNA molecule.
 40. The method of claim 38, wherein different RNA molecules encode the human ORF1p or functional fragment thereof and the human ORF2p or functional fragment thereof.
 41. The method of claim 36, wherein integrating comprises integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA at a poly T site by endonuclease activity of an endonuclease domain of the human ORF2p or a functional fragment thereof.
 42. The method of claim 41, wherein the poly T site comprises the sequence TTTTTA.
 43. The method of claim 31, wherein the isolated RNA molecule comprises homology arms complementary to the target site in the genomic DNA.
 44. The method of claim 31, wherein integrating comprises integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into non-ribosomal genomic DNA of the target human cell or integrating the DNA sequence encoding the exogenous human therapeutic polypeptide into the genomic DNA at a locus that is not an rDNA locus.
 45. The method of claim 31, comprising contacting the target human cell with (i) one or more siRNAs and/or (ii) an RNA guide sequence or a polynucleic acid encoding the RNA guide sequence, and wherein the siRNA or the RNA guide sequence targets a target site of the genomic DNA and the DNA sequence encoding the exogenous human therapeutic polypeptide is integrated into the genomic DNA at the target site of the genomic DNA.
 46. The method of claim 31, wherein the isolated RNA molecule has a total length of from 3 kb to 20 kb.
 47. The method of claim 31, wherein contacting comprises administering the isolated RNA molecule to a human subject.
 48. The method of claim 31, wherein the exogenous human therapeutic polypeptide is selected from the group consisting of a ligand, an antibody, a receptor, an enzyme, a transport protein, a structural protein, a hormone, a contractile protein, a storage protein and a transcription factor.
 49. The method of claim 48, wherein the exogenous human therapeutic polypeptide is a receptor selected from the group consisting of a chimeric antigen receptor (CAR) and a T cell receptor (TCR).
 50. The method of claim 49, wherein the exogenous human therapeutic polypeptide is a receptor selected from the group consisting of a chimeric antigen receptor (CAR) and a T cell receptor (TCR), and wherein the isolated RNA molecule is formulated in a pharmaceutical composition for systemic administration to a human subject.
 51. The method of claim 31, wherein the isolated RNA molecule comprises a 5′ UTR sequence, a 3′ UTR sequence and a poly A sequence; wherein: (i) the 5′ UTR sequence is upstream of the human mobile genetic element, (ii) the 3′ UTR sequence is downstream of the human insert RNA sequence; (iii) the 3′ UTR is upstream of the poly A sequence; wherein the 5′ UTR sequence, the 3′ UTR sequence or the poly A sequence comprises a binding site for the human ORF2p or the functional fragment thereof.
 52. The method of claim 31, wherein the RNA sequence that is a reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide comprises an expression cassette comprising an RNA sequence that is a reverse complement of a promoter sequence, an RNA sequence that is a reverse complement of a 5′ UTR sequence, an RNA sequence that is a reverse complement of a 3′ UTR sequence and an RNA sequence that is a reverse complement of a poly A sequence; wherein (i) the RNA sequence that is a reverse complement of a promoter sequence is downstream of the RNA sequence that is a reverse complement of a 5′ UTR sequence, (ii) the RNA sequence that is a reverse complement of a 5′ UTR sequence is downstream of the RNA sequence that is a reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide, (iii) the RNA sequence that is a reverse complement of a 3′ UTR sequence is upstream of the RNA sequence that is a reverse complement of the DNA sequence encoding the exogenous human therapeutic polypeptide, and (iv) the RNA sequence that is a reverse complement of a poly A sequence is upstream of the RNA sequence that is a reverse complement of a 3′ UTR sequence and downstream of the human mobile genetic element.
 53. The method of claim 31, wherein the isolated RNA molecule comprises a 5′ UTR sequence and a 3′ UTR sequence, wherein (a) the 5′ UTR comprises a 5′ UTR from LINE-1; and/or (b) the 3′ UTR comprises a 3′ UTR from LINE-1.
 54. The method of claim 31, wherein the DNA sequence encoding the exogenous human therapeutic polypeptide does not comprise introns.
 55. The method of claim 31, wherein the isolated RNA molecule comprises a sequence encoding a nuclease domain, wherein the nuclease domain is a nuclease domain from megaTAL, TALEN, Cas9, Cash, Cas7, or Cas8; wherein the nuclease domain is not from ORF2p.
 56. The method of claim 55, wherein the isolated RNA molecule comprises a sequence encoding the nuclease domain, wherein the nuclease domain does not have nuclease activity or comprises a mutation that reduces activity of the nuclease domain compared to the nuclease domain without the mutation.
 57. The method of claim 31, wherein the exogenous human therapeutic polypeptide is expressed in at least 10% of the cells in the target human cell population.
 58. The method of claim 36, wherein the human ORF2p or the functional fragment thereof is a modified human ORF2p that lacks endonuclease activity or has reduced endonuclease activity compared to a wild type human ORF2p.
 59. The method of claim 31, wherein the isolated RNA molecule (i) is formulated in a nanoparticle selected from the group consisting of a lipid nanoparticle and a polymeric nanoparticle; and/or (ii) comprises a glycosylated RNA molecule, a circular RNA molecule or a self-replicating RNA molecule.
 60. The method of claim 31, wherein the polypeptide encoded by the RNA sequence of the human mobile genetic element comprises a sequence with at least 90% sequence identity to SEQ ID NO:
 55. 